High resolution systems, kits, apparatus, and methods for screening microorganisms and other high throughput microbiology applications

ABSTRACT

A microfabricated device defining a high density array of microwells is described for cultivating cells from a sample. The device may be incubated to grow a plurality of cells, which may be split into an analysis portion and a reserve portion. Methods are provided for screening a biological entity of interest using the microfabricated device, for example, for screening phosphate solubilizing bacteria or other bacteria producing acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Nonprovisional patentapplication Ser. No. 15/135,377, filed Apr. 21, 2016, which claims thebenefit of U.S. Provisional Patent Application No. 62/299,088 filed onFeb. 24, 2016, U.S. Provisional Patent Application No. 62/292,091 filedon Feb. 5, 2016, and U.S. Provisional Patent Application No. 62/150,677filed on Apr. 21, 2015. This application also claims the benefit of U.S.Provisional Patent No. 62/410,337 filed Oct. 19, 2016. The disclosure ofeach of these prior-filed applications is incorporated herein byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

This application includes a Sequence Listing which is being submitted inASCII format via EFS-Web, named “GALT_009_US_ST25.txt,” which is 3 KB insize and created on Oct. 17, 2017. The contents of the Sequence Listingare incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates generally to innovations in microbiology,microfabrication, chemistry, optics, robotics, and informationtechnology. More specifically, the present disclosure relates tosystems, apparatus, kits, and methods for high throughput cultivation,screening, isolation, sampling, and/or identification of biologicalentities and/or nutrients.

BACKGROUND

Traditional techniques and tools for cultivating biological entitiesfrom environmental and other samples are often slow, laborious, andexpensive. Even with these techniques and tools, often cells and otherbiological entities still defy all attempts at culture, resulting inmissed information and/or product opportunities. Likewise, the screeningof a population of biological entities for a particular metabolite,enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway,gene, adaptation, capability, and/or therapeutic benefit is challenging,requiring complex and expensive methods. For example, microbes live inextremely high-risk environments. To survive, microbes have developedamazing sets of biochemical tools, including novel enzymes, uniquemetabolites, innovative genetic pathways, and strategies formanipulating their environment and their microbial neighbors—powerfulsolutions that could lead to new insights and products ranging fromlife-saving antibiotics to fertilizers that improve food production andsecurity.

SUMMARY

The present disclosure provides microbiology systems, apparatus, kits,and methods for streamlining the cultivation workflow, supporting highthroughput screening, and/or developing new insights and products inaccordance with some embodiments. For example, an apparatus may comprisea microfabricated device for receiving a sample comprising one or morecells. The microfabricated device defines a high density array ofmicrowells (sometimes referred to as “wells” for simplicity) forcultivating one or more cells.

In some embodiments, a device with a high density array of microwellsfor cultivating cells may be provided. In further embodiments, a set ofunique tags may be provided for disposal into each microwell or some ofthe microwells. Alternatively, the device may be provided with theunique tags already seeded into each microwell or some of themicrowells. The device and/or the unique tags may be provided alone orpart of a kit. For example, a kit may include a microfabricated devicefor receiving a sample comprising one or more cells, the microfabricateddevice defining a high density array of microwells for cultivating thecell(s). A kit also may include a set of unique tags for disposing inthe microwells so as to identify the specific microwell in which aspecies of cell is cultivated. One or more unique tags may be disposedin each microwell of the high density array of microwells. A kit furthermay include instructions for seeding microwells with unique tags and/orusing the unique tags to trace cultivated cells back to the specificmicrowell in which they were cultivated.

In some embodiments, a unique tag may include a nucleic acid moleculewith a target-specific nucleotide sequence for annealing to a targetnucleic acid fragment of a cell or a particular species of cell (e.g.,an organism) that may be present in a microwell and a location-specificnucleotide sequence for identifying the microwell itself. More than oneunique tag may be disposed in a particular microwell such that more thanone location-specific nucleotide sequence is present in the particularmicrowell. That is, the location-specific nucleotide sequences may befound in more than one microwell (e.g., predetermined to identify adimension of microwells in the high density array of microwells), andthe unique tags may be multi-dimensional. For example, a set of uniquetags may be two-dimensional, including tags with a firstlocation-specific nucleotide sequence predetermined to identify a firstdimension of microwells (e.g., a row) in a high density array ofmicrowells and tags with a second location-specific nucleotide sequencepredetermined to identify a second dimension of microwells (e.g., acolumn) in the high density array of microwells. Together, the firstlocation-specific nucleotide sequence and the second location-specificnucleotide sequence identify a specific microwell of the high densityarray of microwells. Thus, the combination of unique tags in aparticular microwell operates to identify that microwell.

In some embodiments, one or more nutrient may be provided for growingand/or screening cells within each or some of the microwells of a deviceincluding a high density array of microwells for cultivating cells. Oneor more nutrients may be provided for disposal in all or some of themicrowells (e.g., directly in a microwell, through a membrane, and/or ona permeable or semi-permeable membrane. Alternatively, the device may beprovided with one or more nutrients already disposed in each microwellor some of the microwells. A nutrient may be provided alone, as part ofa medium, and/or as part of a kit. A kit may include instructions forproviding one or more nutrients to cells being cultivated withinspecific microwells in the device. For example, a kit further mayinclude at least one nutrient for cultivating the at least one speciesof the at least one cell. The at least one nutrient for cultivating theat least one species of the at least one cell may include or be acomponent of an extract from a natural environment of the at least onespecies of the at least one cell, a medium derived from the naturalenvironment of the at least one species of the at least one cell, amedium formulated to resemble the natural environment of the at leastone species of the at least one cell, a selective medium to cultivateonly the at least one species of the at least one cell, and/or adifferential medium to distinguish the at least one species of the atleast one cell. The natural environment of the at least one species ofthe at least one cell may be a biological tissue, a biological product,a microbial suspension, air, soil, sediment, living organic matter,forage, petroleum, sewage, and/or water.

In some embodiments, a membrane may be provided for keeping cells withineach or some of the microwells (e.g., preventing cross-contamination) ofa device including a high density array of microwells for cultivatingcells. A membrane may be applied to seal some or all of the microwells.Alternatively, the device may be provided with the membrane alreadysealed over some or all of the microwells. A membrane may be, forexample, impermeable, permeable only by gas, or allow for diffusion ofone or more nutrients into one or more microwells. The device and/or themembrane may be provided alone or as part of a kit. For example, a kitmay include a microfabricated device for receiving a sample comprisingat least one cell. A kit also may include a membrane to retain the atleast one cell in the at least one microwell of the high density arrayof microwells after the sample is loaded on the microfabricated device.A kit further may include instructions for loading microwells with asample and/or using the membrane keep cultivated cells within thespecific microwells in which they were cultivated.

In some embodiments, steps are provided for cultivating cells using adevice with a high density array of microwells for cultivating cells.For example, a method may include obtaining a microfabricated devicedefining a high density array of microwells for cultivating at least onecell from a sample, which may include disposing at least one unique tagin at least one microwell of the high density array of microwells,loading the device with a sample including one or more cells, and/orapplying a membrane to all or a portion of the device to retain thecells in their respective microwells. In some embodiments, a sample isprepared before loading. For example, a sample may be diluted such thatno more than one cell is disposed in each microwell of a preponderanceof the high density array of microwells. A method also may includeincubating a device to grow a plurality of cells from the at least onecell in the at least one microwell of the high density array ofmicrowells.

In some embodiments, steps are disclosed for replicating and/orsplitting cells cultivated in a high density array of microwells suchthat a portion of the cells may be modified and/or destroyed foranalysis (e.g., amplified and sequenced) whereas the remaining cells maybe reserved for future sampling and/or use based on the informationgleaned from the analysis. For example, a method may include splitting aplurality of cells in the at least one microwell of the high densityarray of microwells into a first portion of the plurality of cells and asecond portion of the plurality of cells. The at least one unique tagmay remain with at least the first portion of the plurality of cells.Splitting the plurality of cells in the at least one microwell of thehigh density array of microwells may include removing the membrane fromthe at least one microwell of the high density array of microwells suchthat some of the cells remain in the at least one microwell of the highdensity array of microwells and some of the cells remain on themembrane. Alternatively, a membrane may be temporarily peeled back toallow sampling or picked through (i.e., punctured) for sampling.

In some embodiments, steps are disclosed for analyzing all or some ofthe cells cultivated in a high density array of microwells. For example,a method may include analyzing a first portion of a plurality of cellsto determine at least one species of interest. The cells to be analyzedmay be in the same microwell in which they were cultivated, attached toa membrane that was covering the microwell in which they werecultivated, and/or transferred to another location (e.g., a petri dishor a second device with a corresponding high density array ofmicrowells). Analysis may include determining a presence or an absenceof at least one species of interest, which may be selected based on atleast one of a nucleic acid and a gene. For example, a polymerase chainreaction (PCR) may be performed to amplify any of the target nucleicacid fragments present in the cells based on the target-specificnucleotide sequence of the at least one unique tag. Analysis may furthercomprise the step of sequencing any PCR-amplified target nucleic acidfragments using next generation sequencing (NGS) or any other type ofsequencing. Sequencing may be used to identify a species of cell (e.g.,an organism) and/or one or more locations in the original high densityarray of microwells at which the species of cell was cultivated.

In some embodiments, steps are disclosed for identifying and/or locatingone or more microwells in a high density array of microwells in which aparticular species of cell was cultivated. For example, a method mayinclude identifying at least one microwell of the high density array ofmicrowells in which at least one species of interest was cultivatedbased on at least one unique tag. Based on identifying the at least onemicrowell of the high density array of microwells in which the at leastone species of interest was cultivated, a method may include locatingthe at least one species of interest within the second portion of theplurality of cells.

In some embodiments, steps are disclosed for picking or sampling one ormore microwells in a high density array of microwells in which aplurality of cells were cultivated. For example, a method may includepicking at least one cell from a particular microwell or a membranelocation correlated to the particular microwell. Devices and methods aredescribed for picking from more than one microwell simultaneously.

In some embodiments, methods are disclosed for screening cells using adevice with a high density array of microwells for cultivating cells.For example, a method may include screening cells cultivated in a highdensity array of microwells to determine a presence or an absence of aspecies of interest. If present, the method may include picking one ormore cells of the species of interest from one or more microwells inwhich the cells were cultivated. A method may include performing anassay based on a metabolite, a metabolic pathway, an enzyme, a protein,a nucleic acid, a phenotype, a gene, a mutation, an adaptation, and/or acapability of the cells. If unique tags were utilized, a method mayinclude using one or more location-specific nucleotide sequences toidentify one or more microwells in which a species of interest wascultivated. For example, a target nucleic acid fragment may be presentin the species of interest, the target nucleic acid fragment includinggenetic code relating to a metabolite, a metabolic pathway, an enzyme, aprotein, a nucleic acid, a phenotype, a gene, a mutation, an adaptation,and/or a capability.

In some embodiments, methods are disclosed for determining a relativeabundance and/or an absolute abundance of a species of interest in asample using a device with a high density array of microwells forcultivating cells. For example, a method may include preparing andloading a sample into microwells with a set of unique tags disposedtherein, the sample prepared such that no more than one cell of the atleast one cell is disposed in each microwell of a preponderance of thehigh density array of microwells. Following cultivation, a method mayinclude using the tags to determine a first number of microwells with atleast one cell, a second number of microwells with the species ofinterest, and based thereupon, a relative abundance of the at least onespecies of interest in the sample. A method may further includedetermining a total number of cells from the sample and, based on thetotal number and a relative abundance of at least one species ofinterest in the sample, an absolute abundance of the at least onespecies of interest in the sample.

More generally, systems, apparatus, kits, and methods are disclosed forcultivating a biological entity from a sample using a microfabricateddevice defining a high density array of experimental units. A biologicalentity may be an organism (e.g., a microorganism such as bacteria orfungi), a cell (e.g., a eukaryotic cell), a cell component, a cellproduct, and a virus. In some embodiments, more than one biologicalentity may be present in a sample, loaded on the device, and/or loadedin an experimental unit. An experimental unit may include one or morebiological entities and/or one or more unique tags to identify spatialinformation relating to the particular experimental unit. A unique tagmay include a binding molecule, such as a protein, a nucleic acid, acarbohydrate, a lipid, and/or a drug. For example, a unique tag mayinclude a target-specific component for binding to a biomolecule presentin a biological entity of interest. A unique tag also may include alocation-specific component for identifying spatial information relatingto the at least one experimental unit. The device may be incubated togrow a plurality of biological entities in the high density array ofexperimental units. One or more cultivated biological entities may beanalyzed to identify one or more species and/or spatial informationrelating to one or more experimental units in which the entities werecultivated based on the at least one unique tag.

In some embodiments, a method of screening for at least one biologicalentity (e.g., bacteria cells, eukaryotic cells) in a sample is provided.The method employs a microfabricated device as described herein that hasa top surface defining an array of microwells. At least one microwell ofthe array of microwells is loaded with: (a) at least one cell from asample; (b) an indicator substance; and (c) an amount of a nutrient. Thecell, the indicator substance, and the nutrient can be loadedsequentially, in any order, or any of their combinations can be loadedsequentially of simultaneously. A membrane is applied to themicrofabricated device to retain the at least one cell in the at leastone microwell. The microfabricated device is incubated at predeterminedconditions for a duration of time to grow a plurality of cells from theat least one cell in the at least one microwell. An optical property,e.g., color, fluorescence, phosphorescence, or luminescence of the atleast one microwell can be evaluated. This evaluation can be done at thecompletion of the incubation, and/or at multiple points in time, e.g.,after loading the microwell contents but before incubation, and afterthe incubation has begun but has not finished, etc. Based on the opticalproperty evaluated or the comparison across multiple times of evaluationof the optical property, a presence or absence of at least onebiological entity of interest in the at least one microwell can bedetermined.

In some embodiments, the optical property of the at least one microwellcorrelates to the amount or form of the indicator substance.

In some embodiments, the indicator substance comprises an inorganicphosphate salt that is insoluble in water. In some of these embodiments,the biological entity of interest can comprise a microorganism thatsolubilizes the inorganic phosphate salt.

In some embodiments, the indicator can be a pH sensitive dye. Forexample, the pH sensitive dye can change color in a visible light rangewithin a selected range of pH. Alternatively, the pH sensitive dye canbe photoluminescent (fluorescent or phosphorescent) in a first range ofpH and not photoluminescent in a second, different range of pH, or a dyewhose photoluminescence changes at different pH.

In some embodiments, the at least one biological entity of interest cancomprise a microorganism that produces an acid.

In some embodiments, measuring the optical property comprises taking aplurality of measurements of the optical property at different timesduring the course of the incubation.

In some embodiments, the method further comprises transferring at leastsome of the plurality of cells after incubation to a target location ifa biological entity of interest is determined to be present in the atleast one microwell.

In some embodiments, the at least one microwell includes a plurality ofmicrowells, and loading the at least one cell comprises loading intoeach of the plurality of microwells, on average, one cell.

In some embodiments, each microwell of the array of microwells has adiameter of about 25 μm to about 500 μm. In some embodiments, thesurface density of the array of microwells is at least 750 microwellsper cm².

In some embodiments, evaluating the optical property comprises: (a)measuring the optical property of the at least one microwell after theat least one cell, the indicator substance, and the nutrient have beenloaded and before incubation; (b) measuring the optical property of theat least one microwell after incubation; and (c) comparing the measuredoptical property before incubation and after incubation.

In some embodiments, a method of screening for at least one biologicalentity of interest in a sample using a microfabricated device having atop surface defining an array of microwells, is provided. The methodcomprises: loading into at least one microwell of the array ofmicrowells: (a) at least one cell from the sample; (b) an indicatorsubstance; and (c) an amount of a nutrient; applying a membrane to themicrofabricated device to retain the at least one cell in the at leastone microwell; incubating the microfabricated device at predeterminedconditions for a duration of time to grow a plurality of cells from theat least one cell in the at least one microwell; and determining apresence or absence of at least one biological entity of interest in theat least one microwell based on a change of the indicator substanceafter incubation.

In some embodiments, a method of screening for at least one biologicalentity of interest in a sample using a microfabricated device having atop surface defining an array of microwells, is provided. The methodcomprises: depositing an indicator substance into each of a plurality ofmicrowells of the array of microwells; loading the sample onto themicrofabricated device such that at least one microwell of the pluralityof microwells receives at least one cell from the sample and an amountof a nutrient; applying a membrane to the microfabricated device toretain the received at least one cell and the nutrient in each of theplurality of microwells; incubating the microfabricated device atpredetermined conditions for a duration of time; and determining apresence or absence of at least one microorganism biological entity ofinterest in the at least one microwell of the plurality of microwellsbased on a measurement of an optical property of the at least onemicrowell.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

Other systems, processes, and features will become apparent to thoseskilled in the art upon examination of the following drawings anddetailed description. It is intended that all such additional systems,processes, and features be included within this description, be withinthe scope of the present invention, and be protected by the accompanyingclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings primarily are forillustrative purposes and are not intended to limit the scope of theinventive subject matter described herein. The drawings are notnecessarily to scale; in some instances, various aspects of theinventive subject matter disclosed herein may be shown exaggerated orenlarged in the drawings to facilitate an understanding of differentfeatures. In the drawings, like reference characters generally refer tolike features (e.g., functionally similar and/or structurally similarelements).

FIG. 1 is a perspective view illustrating a microfabricated device orchip in accordance with some embodiments.

FIGS. 2A-2C are top, side, and end views, respectively, illustratingdimensions of microfabricated device or chip in accordance with someembodiments.

FIGS. 3A and 3B are exploded and top views, respectively, illustrating amicrofabricated device or chip in accordance with some embodiments.

FIGS. 4A and 4B are diagrams illustrating a membrane in accordance withsome embodiments. FIG. 4C is an image of a membrane surface withimpressions formed from contact with an array of wells in accordancewith some embodiments.

FIG. 5A is a flowchart illustrating a method for isolating cells from asample in accordance with some embodiments. FIG. 5B is a diagramillustrating a method for isolating cells from a soil sample inaccordance with some embodiments.

FIG. 6 is a flowchart illustrating a method for isolating andcultivating cells from a sample in accordance with some embodiments.

FIG. 7 is a diagram illustrating a method for isolating and cultivatingcells from a complex sample in accordance with some embodiments. Panel716 shows the output: isolated strains of cultivated cells (SEQ ID NOs:2-6).

FIGS. 8A-8C are diagrams illustrating picking by one pin or multiplepins in accordance with some embodiments.

FIGS. 9A-9D are images demonstrating picking of a well in accordancewith some embodiments.

FIGS. 10A-10D are diagrams illustrating a tool for picking a chip inaccordance with some embodiments.

FIG. 11 is an image of a well that has been picked through a thin layerof agar, illustrating picking through a membrane or sealing layer inaccordance with some embodiments.

FIG. 12 is a diagram illustrating a cross-section of a chip 1200 inaccordance with some embodiments.

FIG. 13 is a flowchart illustrating methods for screening in accordancewith some embodiments.

FIG. 14 is a diagram illustrating a screening method in accordance withsome embodiments.

FIG. 15 is a series of images illustrating a screening example inaccordance with some embodiments.

FIGS. 16A-16C are images illustrating recovery from a screen inaccordance with some embodiments.

FIG. 17A is an exploded diagram illustrating a chip for screening inaccordance with some embodiments. FIG. 17B is a fluorescence image of achip following screening in accordance with some embodiments. FIG. 17Cis an image showing a process of picking a sample from the chipfollowing screening in accordance with some embodiments.

FIG. 18 is a flowchart illustrating a counting method in accordance withsome embodiments.

FIG. 19 is a diagram illustrating a counting method in accordance withsome embodiments. Panel 1916 shows the output: sequences and relativeabundance of cultivated cells (SEQ ID NOs: 2-6).

FIG. 20 is a diagram illustrating an indexing system in accordance withsome embodiments.

FIGS. 21A-21E are diagrams illustrating a chip with well-specificchemistries in accordance with some embodiments.

FIGS. 22A and 22B are images of a chip showing result of ahigh-throughput screening, in accordance with some embodiments.

FIG. 23 is a microscopic image showing an aliquot of a two-componentmixture loaded onto a chip, in accordance with some embodiments.

FIG. 24A shows a composite image which was stitched together from aseries of images taken from the inverted microscope of the chip (withthe reservoir and membrane in place). FIG. 24B is a close-up image takenfrom the inverted microscope showing some microwells with growth andsome microwells empty.

FIG. 25 shows microscopy images of individual microwells as inspectedfor pure cultures (SC, AC, and mixture thereof).

FIGS. 26A and 26B show images of pure cultures of SC (in FIG. 26A) andpure cultures of AC (in FIG. 26B).

FIG. 27 shows gel electrophoresis result for NGS library compiled froman example test according to some embodiments. Lane 1, amplicon frommock community gDNA; lane 2, amplicon from mock community gDNA; lane 3,negative control; lane 4, DNA ladder.

FIG. 28 shows classification of NGS data collected from an example testaccording to some embodiments.

FIG. 29 shows gel electrophoresis result of PCR amplicon from an exampletest according to some embodiments. Lane 1: Ladder. Lanes 2-4: mockcommunity bacteria 16S rRNA amplicon. Lane 5 PCR negative control.

FIG. 30 illustrates a procedure of using dual index for bacteria isolatelocation identification according to some embodiments.

FIG. 31 shows NGS data analysis result for each well from a single 2,500microwells chip according to some embodiments.

FIGS. 32A-32D show microscopic images of microwells of a screening assayfor phosphate solubilizing microorganism according to some embodiments.

DETAILED DESCRIPTION

The present disclosure relates generally to systems, kits, apparatus,and methods for isolation, culturing, adaptation, sampling, and/orscreening of biological entities and/or nutrients. A microfabricateddevice (or a “chip”) is disclosed for receiving a sample comprising atleast one biological entity (e.g., at least one cell). The term“biological entity” may include, but is not limited to, an organism, acell, a cell component, a cell product, and a virus, and the term“species” may be used to describe a unit of classification, including,but not limited to, an operational taxonomic unit (OTU), a genotype, aphylotype, a phenotype, an ecotype, a history, a behavior orinteraction, a product, a variant, and an evolutionarily significantunit.

A cell may be Archaea, Bacteria, or Eukaryota (e.g., fungi). Forexample, a cell may be a microorganism, such as an aerobic, anaerobic,or facultative aerobic microorganisms. A virus may be a bacteriophage.Other cell components/products may include, but are not limited to,proteins, amino acids, enzymes, saccharides, adenosine triphosphate(ATP), lipids, nucleic acids (e.g., DNA and RNA), nucleosides,nucleotides, cell membranes/walls, flagella, fimbriae, organelles,metabolites, vitamins, hormones, neurotransmitters, and antibodies.

A nutrient may be defined (e.g., a chemically defined or syntheticmedium) or undefined (e.g., a basal or complex medium). A nutrient mayinclude or be a component of a laboratory-formulated and/or acommercially manufactured medium (e.g., a mix of two or more chemicals).A nutrient may include or be a component of a liquid nutrient medium(i.e., a nutrient broth), such as a marine broth, a lysogeny broth(e.g., Luria broth), etc. A nutrient may include or be a component of aliquid medium mixed with agar to form a solid medium and/or acommercially available manufactured agar plate, such as blood agar.

A nutrient may include or be a component of selective media. Forexample, selective media may be used for the growth of only certainbiological entities or only biological entities with certain properties(e.g., antibiotic resistance or synthesis of a certain metabolite). Anutrient may include or be a component of differential media todistinguish one type of biological entity from another type ofbiological entity or other types of biological entities by usingbiochemical characteristics in the presence of specific indicator (e.g.,neutral red, phenol red, eosin y, or methylene blue).

A nutrient may include or be a component of an extract of or mediaderived from a natural environment. For example, a nutrient may bederived from an environment natural to a particular type of biologicalentity, a different environment, or a plurality of environments. Theenvironment may include, but is not limited to, one or more of abiological tissue (e.g., connective, muscle, nervous, epithelial, plantepidermis, vascular, ground, etc.), a biological fluid or otherbiological product (e.g., amniotic fluid, bile, blood, cerebrospinalfluid, cerumen, exudate, fecal matter, gastric fluid, interstitialfluid, intracellular fluid, lymphatic fluid, milk, mucus, rumen content,saliva, sebum, semen, sweat, urine, vaginal secretion, vomit, etc.), amicrobial suspension, air (including, e.g., different gas contents),supercritical carbon dioxide, soil (including, e.g., minerals, organicmatter, gases, liquids, organisms, etc.), sediment (e.g., agricultural,marine, etc.), living organic matter (e.g., plants, insects, other smallorganisms and microorganisms), dead organic matter, forage (e.g.,grasses, legumes, silage, crop residue, etc.), a mineral, oil or oilproducts (e.g., animal, vegetable, petrochemical), water (e.g.,naturally-sourced freshwater, drinking water, seawater, etc.), and/orsewage (e.g., sanitary, commercial, industrial, and/or agriculturalwastewater and surface runoff).

A microfabricated device may define a high density array of microwellsfor cultivating the at least one biological entity. The term “highdensity” may refer to a capability of a system or method to distribute anumber of experiments within a constant area. For example, amicrofabricated device comprising a “high density” of experimental unitsmay include about 150 microwells per cm² to about 160,000 microwells ormore per cm², as discussed further herein. Additional examples are shownin TABLE 1.

TABLE 1 Spacing Length of side between Density of of microwellsmicrowells microwells (μm) (μm) (wells/cm2) 500 500 100 100 100 2500 10050 4489 100 10 8281 50 50 10000 50 10 27556 20 10 110889 10 5 444889 5 51000000

A microfabricated device may include a substrate with a series offunctional layers. The series of functional layers may include a firstfunctional layer defining a first array of experimental units (e.g.,wells) and at least one subsequent functional layer defining asubsequent array of experimental units (e.g., microwells) in eachexperimental unit of the preceding functional layer. Each of theexperimental units may be configured to receive and cultivate and/orscreen biological entities and/or nutrients. In particular, systems,kits, apparatus, and methods described herein may be used for automatedand/or high throughput screening of different conditions against a highdensity matrix of cells. For example, systems, kits, apparatus, andmethods described herein may be used to test and compare the effect(s)of one or more different nutrients on the growth of microorganismsand/or screen for metabolites, enzyme activity, mutations, or other cellfeatures.

FIG. 1 is a perspective view illustrating a microfabricated device orchip in accordance with some embodiments. Chip 100 includes a substrateshaped in a microscope slide format with injection-molded features ontop surface 102. The features include four separate microwell arrays (ormicroarrays) 104 as well as ejector marks 106. The microwells in eachmicroarray are arranged in a grid pattern with well-free margins aroundthe edges of chip 100 and between microarrays 104.

FIGS. 2A-2C are top, side, and end views, respectively, illustratingdimensions of chip 100 in accordance with some embodiments. In FIG. 2A,the top of chip 100 is approximately 25.5 mm by 75.5 mm. In FIG. 2B, theend of chip 100 is approximately 25.5 mm by 0.8 mm. In FIG. 2C, the sideof chip 100 is approximately 75.5 mm by 0.8 mm.

After a sample is loaded on a microfabricated device, a membrane may beapplied to at least a portion of a microfabricated device. FIG. 3A is anexploded diagram of the microfabricated device 300 shown from a top viewin FIG. 3B in accordance with some embodiments. Device 300 includes achip with an array of wells 302 holding, for example, soil microbes. Amembrane 304 is placed on top of the array of wells 302. A gasket 306 isplaced on top of the membrane 304. A polycarbonate cover 308 with fillholes 310 is placed on top of the gasket 306. Finally, sealing tape 312is applied to the cover 308.

A membrane may cover at least a portion of a microfabricated deviceincluding one or more experimental units, wells, or microwells. Forexample, after a sample is loaded on a microfabricated device, at leastone membrane may be applied to at least one microwell of a high densityarray of microwells. A plurality of membranes may be applied to aplurality of portions of a microfabricated device. For example, separatemembranes may be applied to separate subsections of a high density arrayof microwells.

A membrane may be connected, attached, partially attached, affixed,sealed, and/or partially sealed to a microfabricated device to retain atleast one biological entity in the at least one microwell of the highdensity array of microwells. For example, a membrane may be reversiblyaffixed to a microfabricated device using lamination. A membrane may bepunctured, peeled back, detached, partially detached, removed, and/orpartially removed to access at least one biological entity in the atleast one microwell of the high density array of microwells.

A portion of the population of cells in at least one experimental unit,well, or microwell may attach to a membrane (via, e.g., adsorption). Ifso, the population of cells in at least one experimental unit, well, ormicrowell may be sampled by peeling back the membrane such that theportion of the population of cells in the at least one experimentalunit, well, or microwell remains attached to the membrane.

FIGS. 4A and 4B are diagrams illustrating a membrane in accordance withsome embodiments. FIG. 4A shows a side view of a chip 400 defining anarray of wells filled with content and a membrane 402 sealed on chip 400over the array of wells, such that the surface of membrane 402 that wasin contact with chip 400, when peeled off chip 400, has impressions ofeach of the wells with samples of the well contents attached (e.g.,stuck) thereto, as shown in FIG. 4B. FIG. 4C is an image of a membranesurface with impressions formed from contact with an array of wells inaccordance with some embodiments.

A membrane may be impermeable, semi-permeable, selectively permeable,differentially permeable, and/or partially permeable to allow diffusionof at least one nutrient into the at least one microwell of a highdensity array of microwells. For example, a membrane may include anatural material and/or a synthetic material. A membrane may include ahydrogel layer and/or filter paper. In some embodiments, a membrane isselected with a pore size small enough to retain at least some or all ofthe cells in a microwell. For mammalian cells, the pore size may be afew microns and still retain the cells. However, in some embodiments,the pore size may be less than or equal to about 0.2 μm, such as 0.1 μm.Membrane diameters and pore sizes depend on the material. For example, ahydrophilic polycarbonate membrane may be utilized, for which thediameter may range from about 10 mm to about 3000 mm, and the pore sizemay range from about 0.01 μm to about 30.0 μm. An impermeable membranehas a pore size approaching zero. In embodiments with an impermeablemembrane, any nutrients must be provided in a microwell prior to beingsealed with the membrane. A membrane that is gas permeable but notliquid permeable may allow oxygen into a microwell and carbon dioxideout of the microwell. The membrane may have a complex structure that mayor may not have defined pore sizes. However, the pores may be on ananometer scale. Other factors in selecting a membrane may include cost,ability to seal, and/or ability to sterilize.

A substrate may define an array of microchannels extended from a firstsurface to a second surface opposite the first surface. A microchannelmay have a first opening in the first surface and a second opening inthe second surface. A first membrane may be applied to at least aportion of the first surface such that at least some of the populationof cells in at least one microchannel attach to the first membrane. Asecond detachable membrane may be applied to at least a portion of thesecond surface such that at least some of the population of cells in atleast one microchannel attach to the second membrane. The population ofcells in the at least one microchannel is sampled by peeling back thefirst membrane such that the at least some of the population of cells inthe at least one microchannel remain attached to the first membraneand/or the second membrane such that the at least some of the populationof cells in the at least one microchannel remain attached to the secondmembrane.

The term “high throughput” may refer to a capability of a system ormethod to enable quick performance of a very large number of experimentsin parallel or in series. An example of a “high throughput” system mayinclude automation equipment with cell biology techniques to prepare,incubate, and/or conduct a large number of chemical, genetic,pharmacological, optical, and/or imaging analyses to screen one or morebiological entities for at least one of a metabolite, an enzyme, aprotein, a nucleic acid, a phenotype, a mutation, a metabolic pathway, agene, an adaptation, and a capability, as discussed herein. According tosome embodiments, “high throughput” may refer to simultaneous or nearsimultaneous experiments on a scale ranging from at least about 96experiments to at least about 10,000,000 experiments.

Systems, kits, apparatus, and methods disclosed herein may be used forhigh throughput screening of different conditions against a matrix ofbiological entities (e.g., cells). A “wells-within-wells” concept may beimplemented by manufacturing (e.g., microfabricating) a substrate orchip to have multiple levels of functional layers to whatever level isrequired or desired (i.e., wells within wells within wells within wells,etc.). A first functional layer may define an array of experimentalunits (e.g., wells). Each of the experimental units presents a secondfunctional layer by defining a subsequent array of experimental units(e.g., microwells). This enables multiple experiments or tests to beperformed at the same time on a single chip, thus enabling highthroughput operation.

For example, in FIGS. 3A and 3B, gasket 306 is placed on top of membrane304, which is applied to an array of wells 302 on a microfabricateddevice 300 in accordance with some embodiments. Gasket 306 has only oneopening. However, in further embodiments, multiple smaller gaskets witha smaller opening or a single gasket with more than one smaller openingmay be placed on top of a device (either with or without a membrane),thereby forming a functional layer or an array of larger experimentalunits with a subsequent functional layer or subsequent array ofexperimental units (e.g., wells 302) located therein.

With multiple levels of functional layers, more than one nutrient ornutrient formulation, for example, can be tested simultaneously or nearsimultaneously. The same format may be used, for example, to screen formetabolites or specific capabilities of cells or to wean microorganismsfrom environmentally derived nutrients to other nutrients.

Experimental units are predetermined sites on a surface of amicrofabricated device. For example, a surface of a chip may be designedto immobilize cells in a first array of predetermined sites. Thesepredetermined sites may be wells, microwells, microchannels, and/ordesignated immobilization sites. For example, a surface may bemanufactured to define an array of microwells. The array may be dividedinto sections by defining walls in the substrate or adding walls. Forexample, the surface may be manufactured to first define a first arrayof wells, in which an inner surface of each well, in turn, ismanufactured to define a second array of microwells, microchannels, orimmobilization sites. In another example, the surface may bemanufactured to define an array of microwells, and another substrate(e.g., agar, plastic, or another material) is applied to the surface topartition the surface and the microwells defined thereby. Each well,microwell, microchannel, and/or immobilization site may be configured toreceive and grow at least one cell; however, in use, any given well,microwell, microchannel, or immobilization site may or may not actuallyreceive and/or grow one or more cells. Types of experimental units maybe interchangeable. For example, embodiments herein that expresslydescribe microwells are also intended to disclose embodiments in whichthe microwells are at least in part replaced with microchannels,immobilization sites, and/or other types of experimental units.

One or more portions of a microfabricated device may be selected,treated, and/or coated with a surface chemistry modifier to have aparticular surface chemistry. For example, at least a portion of asubstrate surface may be configured with first surface characteristicsthat repel cells and/or reduce cellular tendency to stick to the surfaceor second surface characteristics that attract cells and/or increasecellular tendency to attach to the surface. Depending on the type oftarget cell, the material and/or coating may be hydrophobic and/orhydrophilic. At least a portion of the top surface of the substrate maybe treated to have first surface characteristics that repel target cellsand/or reduce the tendency of target cells to stick to the surface.Meanwhile, at least a portion of the inner surface of each experimentalunit, well, or microwell may be treated to have second surfacecharacteristics that attract target cells and increase the tendency oftarget cells to occupy the experimental unit, well, or microwell. Asurface of a substrate may have a plurality of portions with differentsurface characteristics.

A surface chemistry modifier may be applied using chemical vapordeposition, electroporation, plasma treatment, and/or electrochemicaldeposition. The surface chemistry modifier may control surfacepotential, Lund potential, zeta potential, surface morphology,hydrophobicity, and/or hydrophilicity. The surface chemistry modifiermay include a silane, a polyelectrolyte, a metal, a polymer, anantibody, and/or a plasma. For example, the surface chemistry modifiermay include octadecyltrichlorosilane. The surface chemistry modifier mayinclude a dynamic copolymer, such as polyoxyethylene (20) sorbitanmonolaurate and/or polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether. The surface chemistrymodifier may include a static copolymer, such as poloxamer 407,poly(L-lysine), and/or a poly(ethylene glycol)-poly(l-lysine) blockcopolymer.

An apparatus for screening different conditions against a matrix ofcells may include a substrate with a surface defining an array ofmicrowells. Sections of the microwell array may be partitioned intosubarrays (e.g., by larger wells or walls). The substrate may bemicrofabricated. Each microwell may receive and grow at least onebiological entity (e.g., cell). The resulting matrix of biologicalentities (e.g., cells) may be a high density matrix of biologicalentities. The first array and/or the second array may be planar,substantially planar, and/or multi-planar (e.g., on a roll).

The term “high resolution” may refer to a capability of a system ormethod to distinguish between a number of available experiments. Forexample, a “high resolution” system or method may select an experimentalunit from a microfabricated device comprising a high density ofexperimental units, in which the experimental unit has a diameter fromabout 1 nm to about 800 μm. A substrate of a microfabricated device orchip may include about or more than 10,000,000 microwells. For example,an array of microwells may include at least 96 locations, at least 1,000locations, at least 5,000 locations, at least 10,000 locations, at least50,000 locations, at least 100,000 locations, at least 500,000locations, at least 1,000,000 locations, at least 5,000,000 locations,or at least 10,000,000 locations.

The surface density of microwells may be from about 150 microwells percm² to about 160,000 microwells per cm² or more. A substrate of amicrofabricated device or chip may have a surface density of microwellsof at least 150 microwells per cm², at least 250 microwells per cm², atleast 400 microwells per cm², at least 500 microwells per cm², at least750 microwells per cm², at least 1,000 microwells per cm², at least2,500 microwells per cm², at least 5,000 microwells per cm², at least7,500 microwells per cm², at least 10,000 microwells per cm², at least50,000 microwells per cm², at least 100,000 microwells per cm², or atleast 160,000 microwells per cm².

The dimensions of a microwell may range from nanoscopic (e.g., adiameter from about 1 to about 100 nanometers) to microscopic or larger.For example, each microwell may have a diameter of about 1 μm to about800 μm, a diameter of about 25 μm to about 500 μm, or a diameter ofabout 30 μm to about 100 μm. A microwell may have a diameter of about orless than 1 μm, about or less than 5 μm, about or less than 10 μm, aboutor less than 25 μm, about or less than 50 μm, about or less than 100 μm,about or less than 200 μm, about or less than 300 μm, about or less than400 μm, about or less than 500 μm, about or less than 600 μm, about orless than 700 μm, or about or less than 800 μm.

A microwell may have a depth of about 500 μm to about 5000 μm, a depthof about 1 μm to about 500 μm, or a depth of about 25 μm to about 100μm. A microwell may have a depth of about 1 μm, about 5 μm, about 10 μm,about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm,about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm,about 1000 μm, about 1,500 μm, about 2,000 μm, about 3,000 μm, or about5,000 μm.

Each microwell may have an opening or cross section having any shape,e.g., round, hexagonal, or square. Each microwell may include sidewalls.For microwells that are not round in their openings or cross sections,the diameter of the microwells described herein refer to the effectivediameter of a circular shape having an equivalent area. For example, fora square shaped microwell having side lengths of 10×10 microns, a circlehaving an equivalent area (100 square microns) has a diameter of 11.3microns. At least one unique location-specific tag, as described furtherbelow, may be disposed in at least one microwell of the high densityarray of microwells to facilitate identification of a species and acorrelation of a species to a specific microwell of the high densityarray of microwells. The at least one unique tag may be disposed and/orpositioned at the bottom of the microwell and/or on at least one side ofthe microwell. The at least one unique tag may include a nucleic acidmolecule with a target-specific nucleotide sequence for annealing to atarget nucleic acid fragment of the at least one biological entity and alocation-specific nucleotide sequence for identifying the at least onemicrowell of the high density array of microwells.

For example, a substrate of a microfabricated device or chip may have asurface with dimensions of about 4 inches by 4 inches. The surface maydefine an array of approximately 100 million microwells. The microwellarray may be partitioned into about 100 subsections by walls and/or thesubstrate may define an array of about 100 wells, with about one millionmicrowells defined within each subsection or well totaling toapproximately 100 million microwells. For a use case of testingdifferent nutrients, microorganisms from an environmental sample may beloaded on the chip such that individual microorganisms or clusters ofmicroorganisms partition into the microwells on the chip, each microwellbeing located at the bottom of a larger well. Each larger well mayinclude an experimental unit such that about 100 different nutrients maybe tested in parallel or in series on the same chip, with each wellproviding up to 1 million test cases.

Target cells may be Archaea, Bacteria, or Eukaryota (e.g., fungi,plants, or animals). For example, target cells may be microorganisms,such as aerobic, anaerobic, and/or facultative aerobic microorganisms.Different nutrients may be tested in parallel or in series on acomposition of target cells to analyze and compare, for instance, growthor other effects on cell population, cell components, and/or cellproducts. A composition of target cells may be screened for a cellcomponent, product, and/or capability, such as one or more of a virus(e.g., a bacteriophage), a cell surface (e.g., a cell membrane or wall),a metabolite, a vitamin, a hormone, a neurotransmitter, an antibody, anamino acid, an enzyme, a protein, a saccharide, ATP, a lipid, anucleoside, a nucleotide, a nucleic acid (e.g., DNA or RNA), aphenotype, a mutation, a metabolic pathway, a gene, and an adaptation.

A composition of cells may include an environmental sample extractand/or a dilutant. The environmental sample extract and/or the dilutantmay include, but is not limited to, one or more of a biological tissue(e.g., connective, muscle, nervous, epithelial, plant epidermis,vascular, ground, etc.), a biological fluid or other biological product(e.g., amniotic fluid, bile, blood, cerebrospinal fluid, cerumen,exudate, fecal matter, gastric fluid, interstitial fluid, intracellularfluid, lymphatic fluid, milk, mucus, rumen content, saliva, sebum,semen, sweat, urine, vaginal secretion, vomit, etc.), a microbialsuspension, air (including, e.g., different gas contents), supercriticalcarbon dioxide, soil (including, e.g., minerals, organic matter, gases,liquids, organisms, etc.), sediment (e.g., agricultural, marine, etc.),living organic matter (e.g., plants, insects, other small organisms andmicroorganisms), dead organic matter, forage (e.g., grasses, legumes,silage, crop residue, etc.), a mineral, oil or oil products (e.g.,animal, vegetable, petrochemical), alcohol, a buffer, an organicsolvent, water (e.g., naturally-sourced freshwater, drinking water,seawater, etc.), and/or sewage (e.g., sanitary, commercial, industrial,and/or agricultural wastewater and surface runoff).

A method may include, prior to applying (e.g., loading) a compositionincluding cells to a microfabricated device, preparing the compositionby combining the cells with an environmental sample extract and/or adilutant. The method further may include liquefying the environmentalsample extract and/or the dilutant. A concentration of cells in acomposition may be adjusted to target distribution of one cell perexperimental unit, well, or microwell.

If a sample contains cells and/or viruses, the cells in the sample maybe lysed after they are applied to a microfabricated device to releasenucleic acid molecules. Cells may be lysed with chemical treatment suchas alkaline exposure, detergents, sonication, enzymatic proteinase K, orlysozyme exposure. Cells may also be lysed by heating.

FIG. 5A is a flowchart illustrating a method for isolating cells from asample in accordance with some embodiments. In step 500, a sample isobtained. In step 502, the sample is homogenized and/or dispersed usingat least one of a physical technique (e.g., blending and/or sonication)and a chemical technique (e.g., chelating agents, detergents, and/orenzymes). In step 504, cells in the homogenized and/or dispersed sampleare separated by density centrifugation using, for example, Nycodenz®non-particulate medium (available from Progen Biotechnik GmbH,Heidelberg, Germany).

FIG. 5B is a diagram illustrating a method for isolating cells from asoil sample in accordance with some embodiments. Panel 506 shows thesoil sample. Panel 508 shows the homogenized and/or dispersed sample ina test tube. Panel 510 shows the sample after centrifugation, separatedinto soluble debris 512, cells 514, insoluble debris 516, and Nycodenz®518.

FIG. 6 is a flowchart illustrating a method for isolating andcultivating cells from a sample in accordance with some embodiments. Instep 600, a sample is obtained. In step 602, at least one cell isextracted from the obtained sample. In step 604, at least one highdensity microwell array of a microfabricated device or chip is loadedwith the at least one extracted cell. Step 604 may include preparing acell concentration with the at least one extracted cell, selecting atleast one nutrient/media, and/or selecting at least one membrane. Instep 606, at least a portion of the microwell array is sealed with theat least one selected membrane to retain the cell concentration with themicrowells. In step 608, the chip is incubated. Step 608 may includeselecting a temperature, determining atmosphere (e.g., aerobic oranaerobic), and/or timing incubation). In step 610, the chip is splitand/or duplicated (using, e.g., a picker), resulting in two portions ofcultivated cells according to methods described herein. For example, theat least one membrane may be peeled off such that a portion of thecultivated cells remain attached or peeled off or punctured to samplethe cultivated cells. In optional step 612, one portion of thecultivated cells is sacrificed for identification. Step 612 may includePCR, sequencing, and/or various data analytics. In step 614, strains ofinterest are identified. Further cultivation, testing, and/oridentification may be performed with, for example, the strains ofinterest and/or the remaining portion of the cultivated cells.

FIG. 7 is a diagram illustrating a method for isolating and cultivatingcells from a complex sample in accordance with some embodiments. Panel700 shows examples of complex samples, specifically a microbiome sample702 and a soil sample 704. In Panel 706, at least one cell is extractedfrom the sample using, for example, the protocol illustrated in FIGS. 5Aand 5B. In Panel 708, the at least one extracted cell (and anyenvironmental extract and/or dilutant) is loaded on a microfabricateddevice or chip with at least one high density microwell array 710. Chip710 and a reagent cartridge 712 may be loaded into an incubator 714. Thereagent may be useful for adding liquid to maintain nutritionalrequirements for growth and/or various screening purposes. Panel 716shows the output: isolated strains of cultivated cells.

To identify the species or taxonomic lineage of cells or microorganismsgrowing in a microwell requires techniques including, but not limitedto, DNA sequencing, nucleic acid hybridization, mass spectrometry,infrared spectrometry, DNA amplification, and antibody binding toidentify genetic elements or other species identifiers. Manyidentification methods and process steps kill the microorganisms andtherefore prevent further cultivation and study of microorganisms ofinterest. To enable both the identification of cells or microorganismswhile enabling subsequent cultivation, study, and further elaboration ofparticular clones of interest, further embodiments are designed forsampling each experimental unit, well, or microwell across a substrateor chip while maintaining the locational integrity and separation ofmicroorganism populations across experimental units, wells, ormicrowells.

A substrate as described above may enable sampling a cell populationusing further systems, kits, apparatus, and methods. For example, apicking device may be applied to a first surface of the substrate. Thedevice may include at least one protrusion facing the first surface. Theat least one protrusion has a diameter less than the opening diameter ofeach microwell, well, or experimental unit. The at least one protrusionmay be inserted into at least one microwell, well, or experimental unitholding a population of cells such that a portion of the population ofcells in the at least one microwell, well, or experimental unit adheresand/or attaches to the at least one protrusion. The sample of thepopulation of cells in the at least one microwell, well, or experimentalunit may be withdrawn by removing the device from the first surface ofthe substrate such that the portion of the population of cells in the atleast one microwell, well, or experimental unit remains adhered and/orattached to the at least one protrusion. Each protrusion may be a pin ora plurality or assembly of pins.

FIGS. 8A-8C are diagrams illustrating picking by one pin or multiplepins in accordance with some embodiments. Chip 800 is provided forinspection via a microscope 802 and picking via picking control device804. In FIG. 8A, picking control device 804 comprises an arm with asingle pin 806. In FIG. 8B, an arm with multiple pins 808 is shown. FIG.8C is a perspective view of the chip during the picking process.

FIGS. 9A-9D are images demonstrating picking of a well in accordancewith some embodiments. In FIG. 9A, the well is full. In FIG. 9B, the pinis moved into position. In FIG. 9C, the well is picked. In FIG. 9D, asample is removed from the well.

FIGS. 10A-10D are diagrams illustrating a tool for picking a chip inaccordance with some embodiments. In FIG. 10A, a tool comprising aplurality of pins is aligned with a chip having a plurality of wells. InFIG. 10B, the tool is lowered such that the pins are dipped into thewells. In FIG. 10C, the pins are shown with samples attached, and thesamples are transferred to a new chip. Alternatively, in FIG. 10D, thetool is flipped such that the samples may be maintained in the toolitself.

FIG. 11 is an image of a well that has been picked through a thin layerof agar, illustrating picking through a membrane or sealing layer inaccordance with some embodiments.

Alternatively, when the at least one protrusion is inserted into the atleast one microwell, well, or experimental unit, a portion of thepopulation of cells in the at least one the at least one microwell,well, or experimental unit is volume displaced up and around the atleast one protrusion such that at least some of the volume displacedportion is above the first surface of the substrate and/or the innersurface of the at least one microwell, well, or experimental unit. Themethod also includes sampling the population of cells in the at leastone microwell by collecting at least some of the volume displacedportion of the population of cells.

A similar picking device may be applied to a second surface opposite thefirst surface of the substrate. The device may include at least oneprotrusion facing the second surface. The at least one protrusion has adiameter about equal to or less than a diameter of at least onemicrowell, well, or experimental unit. The at least one protrusion ispushed against the second surface at a location corresponding to the atleast one microwell, well, or experimental unit holding a population ofcells and/or inserted into the at least one microwell, well, orexperimental unit holding the population of cells such that a portion ofthe population of cells in the at least one microwell, well, orexperimental unit is displaced above the first surface of the substrateand/or the inner surface of the at least one microwell, well, orexperimental unit. The displaced portion of the population of cells maythen be collected. The population of cells may be located on a plug(e.g., a hydrogel or other soft material like agar) in the at least oneexperimental unit, well, or microwell such that when the at least oneprotrusion is at least one of pushed against the second surface andinserted into the at least one microwell, the plug is displaced, therebydisplacing the portion of the population of cells.

The sample of the population of cells from the at least one experimentalunit, well, or microwell may be deposited in a second location. The atleast one protrusion may be cleaned and/or sterilized prior to furthersampling. At least a portion of the at least one protrusion may becomposed of a material, treated, and/or coated with a surface chemistrymodifier for surface characteristics that favor attachment of cells. Theat least one protrusion may be an array of protrusions. Upon applyingthe device to the first surface of the substrate, the array ofprotrusions may be inserted into a corresponding array of experimentalunits, wells, or microwells. The number of protrusions in the array ofprotrusions may correspond to the number of experimental units in thefirst array, the number of microwells in one second array of microwells,or the total number of microwells in the substrate.

Another device for sampling a cell population in a substrate includes atleast one needle and/or nanopipette facing the first surface. The atleast one needle and/or nanopipette has an external diameter less thanthe opening diameter of each microwell and an internal diameter capableof accommodating a target cell diameter. The at least one needle and/ornanopipette is inserted into at least one experimental unit, well, ormicrowell holding a population of cells. The sample of the population ofcells in the at least one experimental unit, well, or microwell iswithdrawn using pressure to pull a portion of the population of cellsfrom the at least one experimental unit, well, or microwell into thedevice.

The sample of the population of cells from the at least one experimentalunit, well, or microwell may be deposited in a second location. The atleast one needle and/or nanopipette may be cleaned and/or sterilizedprior to further sampling. The at least one needle and/or nanopipettemay be an array of needles and/or nanopipettes. Upon applying the deviceto the first surface of the microfabricated substrate, the array ofneedles and/or nanopipettes may be inserted into a corresponding arrayof experimental units, wells, or microwells. The number of needlesand/or nanopipettes in the array of needles and/or nanopipettes maycorrespond to the number of the experimental units in the first array,the number of microwells in one second array of microwells, or the totalnumber of microwells in the substrate.

Another method for sampling a cell population in a substrate includesapplying focused acoustic energy to at least one experimental unit,well, or microwell holding a population of cells in fluid. The focusedacoustic energy may be applied in a manner effective to eject a dropletfrom the at least one microwell, such as, for example, acoustic dropletejection (ADE) (see, e.g., Sackmann et al., “Acoustical Micro- andNanofluidics: Synthesis, Assembly and Other Applications,” Proceedingsof the 4th European Conference on Microfluidics (December 2014)). Thedroplet may include a sample of the population of cells in the at leastone experimental unit, well, or microwell. The droplet may be directedinto a second container or surface or substrate.

A substrate may include at least a first piece including at least aportion of the first surface and a second piece including at least aportion of the second surface. The first piece and the second piece aredetachably connected along at least a portion of a plane parallel to thefirst surface and the second surface. The plane divides the experimentalunits, wells, or microwells. A cell population in at least oneexperimental unit, well, or microwell is sampled by detaching the firstpiece and the second piece such that a first portion of the populationof cells in the at least one experimental unit, well, or microwellremains attached to the first piece and a second portion of thepopulation of cells in the at least one experimental unit, well, ormicrowell remains attached to the second piece.

FIG. 12 is a diagram illustrating a cross-section of a chip 1200 inaccordance with some embodiments. Chip 1200 includes a substratedefining an array of wells 1202 filled with contents 1204. The substratecomprises a first piece 1206 and a second piece 1208. The first piece1206 and the second piece 1208 are detachably connected along a plane1210 parallel to and bisecting the array of wells 1202. When the firstpiece 1206 and the second piece 1208 are detached, the wells 1202 andtheir contents 1204 are divided, resulting in two copies of the contents1204 that preserve both the isolation and the location of the contents1204 on chip 1200. Each microwell, experimental unit, or microchannelmay include a partial barrier that partially separates the microwell,experimental unit, or microchannel into a first portion and a bottomportion such that a cell population is able to grow in both the firstportion and the bottom portion. Prior to sampling the population ofcells, the above methods may include dispersing and/or reducing clumpsof cells in the population of cells. Dispersing and/or reducing clumpsof cells in the population of cells may include, but is not limited to,applying sonication, shaking, and dispension with small particles.

The above methods further may include depositing the sample of thepopulation of cells from the at least one experimental unit, well, ormicrowell in a second location. The second location may be acorresponding array of experimental units, wells, or microwells. Thesecond location may be a single receptacle. The sample of the populationof cells from the at least one experimental unit, well, or microwell maybe maintained for subsequent cultivation. Alternatively, the remainingcells of the population of cells in the at least one experimental unit,well, or microwell may be maintained for subsequent cultivation.

The above methods further may include identifying at least one cell fromthe sample of the population of cells and/or the remaining cells of thepopulation of cells. This may include performing DNA, cDNA, and/or RNAamplification, DNA and/or RNA sequencing, nucleic acid hybridization,mass spectrometry, and/or antibody binding. Alternatively, or inaddition, this may include identifying an experimental unit, well, ormicrowell from which at least one cell originated. Each experimentalunit, well, or microwell may be marked with a unique tag including alocation-specific nucleotide sequence. To identify the experimentalunit, well, or microwell, a location-specific nucleotide sequence may beidentified in the sequencing and/or amplification reaction, and thelocation specific nucleotide sequence may be correlated with the atleast one experimental unit, well, or microwell from which the at leastone cell originated.

A microfabricated device as described above may enable culturing cellsin a sample derived from an environment using further systems, kits,apparatus, and methods. For example, a sample may be applied to thefirst surface of a substrate such that at least one of the cellsoccupies at least one microwell, well, or experimental unit. Asemi-permeable membrane is applied to at least a portion of the firstsurface (e.g., at least a portion of an inner surface of an experimentalunit or well) such that a nutrient can diffuse into the at least onemicrowell, well, or experimental unit. Meanwhile, escape of theoccupying cells from the at least one microwell, well, or experimentalunit is prevented and/or mitigated. A semi-permeable membrane may be,for example, a hydrogel layer. A semi-permeable membrane may bereversibly or irreversibly connected or affixed to the substrate using,for example, lamination. Thus, the occupying cells may be incubated inthe at least one microwell, well, or experimental unit with at least onenutrient. The cells may be gradually transitioned over a period of timefrom at least one nutrient to at least one alternative nutrient ornutrient formulation using progressive partial exchange, therebyundergoing domestication or adaptation.

A first nutrient derived from the environment may be used to incubatethe cells occupying at least one first experimental unit, well, ormicrowell, and a second nutrient derived from the environment may beused to incubate the cells occupying at least one second experimentalunit, well, or microwell. The above methods may include comparing thecells occupying the at least one first experimental unit, well, ormicrowell with the cells occupying at least one second experimentalunit, well, or microwell to analyze the first nutrient and the secondnutrient.

For example, a method may include one or more of the following steps:

-   -   Acquire a chip defining 1000 to 10 million or more microwells        within a number of larger wells or flow cells, each microwell        having a diameter of about 1 μm to about 800 μm and a depth of        about 1 μm to about 800 μm, the chip further having one or more        surface chemistries configured to facilitate the movement of        target microorganisms into the microwells;    -   Apply an environmental sample or a derivative of the        environmental sample to the chip such that any target        microorganisms become located in the microwells;    -   Place one or more semi-permeable filters, hydrogel layers, or        other barriers on the chip such that a barrier is created that        allows nutrients to diffuse into the microwells but prevents        and/or mitigates escape of microorganisms from the microwells;    -   Incubate the chip with at least one nutrient (e.g., derived from        the environment);    -   Gradually change the nutrient source by progressive partial        exchange with at least one alternative nutrient (e.g.,        formulation); and    -   Detect any growth of microorganisms in the microwells.

The target cells may be Archaea, Bacteria, or Eukaryota. Target virusesmay be bacteriophages. When viruses are targeted, the microwells of thechip may also include host cells in which the viruses may grow.Detecting the growth of the occupying cells or viruses may includedetecting a change in biomass (e.g., DNA/RNA/protein/lipid), metabolitepresence or absence, pH, consumption of nutrients, and/or consumption ofgases. Detecting the growth of the occupying cells or viruses mayinclude performing real-time sequential imaging, microscopy, opticaldensity, fluorescence microscopy, mass spectrometry, electrochemistry,amplification (DNA, cDNA, and/or RNA), sequencing (DNA and/or RNA),nucleic acid hybridization, and/or antibody binding.

FIG. 13 is a flowchart illustrating methods for screening in accordancewith some embodiments. In step 1300, a sample is obtained. In step 1302,at least one cell is extracted from the obtained sample. In step 1304,at least one high density microwell array of a microfabricated device orchip is loaded with the at least one extracted cell. Step 1304 mayinclude preparing a cell concentration with the at least one extractedcell, selecting at least one nutrient/media, and/or selecting at leastone membrane. In step 1306, at least a portion of the microwell array issealed with the at least one selected membrane to retain the cellconcentration with the microwells. In step 1308, the chip is incubated.Step 1308 may include selecting a temperature, determining atmosphere(e.g., aerobic or anaerobic), and/or timing incubation). A geneticscreen and/or a functional screen may be performed. In step 1310, agenetic screen is applied to the chip. In step 1312, the chip is splitand/or duplicated (using, e.g., a picker), resulting in two portions ofcultivated cells according to methods described herein. For example, theat least one membrane may be peeled off such that a portion of thecultivated cells remain attached or peeled off or punctured to samplethe cultivated cells. In optional step 1314, one portion of thecultivated cells is sacrificed for identification. Step 1314 may includePCR, sequencing, and/or various data analytics. In step 1316, strains ofinterest are identified. Further cultivation, testing, and/oridentification may be performed with, for example, the strains ofinterest and/or the remaining portion of the cultivated cells.Alternatively, in step 1318, a functional screen is applied to the chip.In step 1320, one or more variables are observed and, as in step 1316,strains of interest are identified.

FIG. 14 is a diagram illustrating a screening method in accordance withsome embodiments. Panel 1400 shows examples of complex samples,specifically a microbiome sample 1402 and a soil sample 1404. In Panel1406, at least one cell is extracted from the sample using, for example,the protocol illustrated in FIGS. 5A and 5B. In Panel 1408, the at leastone extracted cell (and any environmental extract and/or dilutant) isloaded on a microfabricated device or chip with at least one highdensity microwell array 1410. Chip 1410 and a reagent cartridge 1412 maybe loaded into an incubator 1414. The reagent may be useful for addingliquid to maintain nutritional requirements for growth and/or variousscreening purposes. Panel 1416 shows the output: screen results andisolated strains of cultivated cells.

FIG. 15 is a series of images illustrating a screening example inaccordance with some embodiments. The images show portions of a chipwith a membrane and an acid-sensitive layer applied thereon to screenfor low pH. In image 1500, more than 1800, 50 μm microwells are visiblewith nine clear hits 1502. Image 1504 is a magnified view of box 1504,and image 1506 is a magnified view of one of the microwells with a hit1502.

FIGS. 16A-16C are images illustrating recovery from a screen inaccordance with some embodiments. In FIG. 16A, at least one well ispicked using a microscope and a picking device with at least one pin. InFIG. 16B, a pin is removed and incubated in media. In FIG. 16C, growthis visible.

FIG. 17A is an exploded diagram illustrating a chip for screening inaccordance with some embodiments. In FIG. 17A, chip 1700 includes a highdensity array of microwells with, for example, soil microbes in themicrowells. Membrane 1702 is applied to chip 1700. Gasket 1704 isapplied to chip 1700 over membrane 1702. Agar with fluorescent E. colibacteria 1706 is applied to chip 1700 over gasket 1704 and membrane1702. FIGS. 17B and 17C are images illustrating a screening example inaccordance with some embodiments. In this example, the screen is forclearance zones. FIG. 17B is a fluorescence image of a chip, preparedlike chip 1700 in FIG. 17A, following the screen. FIG. 17C is an imageshowing a process of picking a sample from this chip through the agar.

In some embodiments, a location on an apparatus may be correlated with aportion of a sample present at that location, after that portion of thesample (or a part of the portion) is removed from the apparatus. Theapparatus may be or include a microarray. The microarray may comprise aplurality of locations for applying a sample, wherein each location ismarked with a unique tag which may be used to identify the location fromwhich a portion of the sample came, after that portion of the sample isremoved from the microarray.

The disclosure relates to a method of identifying from which location ona microarray a portion of a sample comprising at least one nucleic acidmolecule came, after that portion of the sample is removed from themicroarray, the method comprising the steps of: (a) applying one or moreportions of the sample onto one or more of a plurality of locations onthe microarray, wherein each location is marked with a unique tagcomprising a nucleic acid molecule comprising: (i) a location-specificnucleotide sequence; and (ii) a first target-specific nucleotidesequence; (b) allowing the target nucleic acid molecule found in atleast one portion of the sample to anneal to a tag marking a location;(c) performing primer extension, reverse transcription, single-strandedligation, or double-stranded ligation on the population of annealednucleic acid molecules, thereby incorporating a location-specificnucleotide sequence into each nucleic acid molecule produced by primerextension, reverse transcription, single-stranded ligation, ordouble-stranded ligation; (d) combining the population of nucleic acidmolecules produced in step (c); (e) sequencing the population ofcombined nucleic acid molecules, thereby obtaining the sequence of oneor more location-specific nucleotide sequences; and (f) correlating thesequence of at least one location-specific nucleotide sequence obtainedfrom the population of combined nucleic acid molecules to the locationon the microarray marked with a tag comprising said location-specificnucleotide sequence; thereby identifying from which location on amicroarray a portion of a sample comprising at least one nucleic acidmolecule came. In some embodiments, a sample may include at least onecell and one or more nucleic acid molecules are released from the cellafter step (a) and before step (b). A sample may include at least onecell, and the at least one cell replicates or divides after step (a) andbefore step (b). A portion of the portion of the sample may be removedfrom at least one location before step (b) and said portion of theportion of the sample may be stored in a separate receptacle correlatedto the original location of the portion of the sample on the microarray.The method of correlating or identifying a location may further comprisethe step of amplifying the nucleic acid molecules produced in step (c)or the population of combined nucleic acid molecules produced in step(d). The amplifying step may comprise polymerase chain reactionamplification, multiplexed polymerase chain reaction amplification,nested polymerase chain reaction amplification, ligase chain reactionamplification, ligase detection reaction amplification, stranddisplacement amplification, transcription based amplification, nucleicacid sequence-based amplification, rolling circle amplification, orhyper-branched rolling circle amplification. Additional primers may beadded during an amplification reaction. For example, both 5′ and 3′primers may be needed for a PCR reaction. One of the primers used duringan amplification reaction may be complementary to a nucleotide sequencein the sample.

In some embodiments, a composition including cells and/or viruses may betreated with a nuclease before the composition is applied to amicrofabricated device so that contaminating nucleic acid molecules arenot amplified in subsequent steps.

The sequencing used in the disclosed methods and apparatuses may be anyprocess of obtaining sequence information, including hybridization anduse of sequence specific proteins (for example, enzymes). Sequencing maycomprise Sanger sequencing, sequencing by hybridization, sequencing byligation, quantitative incremental fluorescent nucleotide additionsequencing (QIFNAS), stepwise ligation and cleavage, fluorescenceresonance energy transfer, molecular beacons, TaqMan reporter probedigestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ),wobble sequencing, multiplex sequencing, polymerized colony (POLONY)sequencing (see, e.g., U.S. Patent Application Publication No.2012/0270740, which is incorporated by reference herein in itsentirety); nanogrid rolling circle (ROLONY) sequencing (see, e.g., U.S.Patent Application Publication No. 2009/0018024, which is incorporatedby reference herein in its entirety), allele-specific oligo ligationassay sequencing, or sequencing on a next-generation sequencing (NGS)platform. Non-limiting examples of NGS platforms include systems fromIllumina® (San Diego, Calif.) (e.g., MiSeq™, NextSeq™, HiSeq™, and HiSeqX™), Life Technologies (Carlsbad, Calif.) (e.g., Ion Torrent™), andPacific Biosciences (Menlo Park, Calif.) (e.g., PacBio® RS II).

An organism or species may be identified by comparing the nucleic acidsequence obtained from that organism to various databases containingsequences of organisms. For example, ribosomal RNA sequence data isavailable in the SILVA rRNA database project (Max Planck Institute forMarine Microbiology, Bremen, Germany (www.arb-silva.de); see, e.g.,Quast et al., “The SILVA Ribosomal RNA Gene Database Project: ImprovedData Processing and Web-Based Tools,” 41 Nucl. Acids Res. D590-D596(2013), and Pruesse et al., “SINA: Accurate High-Throughput MultipleSequence Alignment of Ribosomal RNA Genes,” 28 Bioinformatics 1823-1829(2012), both of which are incorporated by reference herein in theirentirety). Other ribosomal RNA sequence databases include the RibosomalDatabase Project (Michigan State University, East Lansing, Mich.(www.rdp.cme.msu.edu); see, e.g., Cole et al., “Ribosomal DatabaseProject: Data and Tools for High Throughput rRNA Analysis” 42 Nucl.Acids Res. D633-D642 (2014), which is incorporated by reference hereinin its entirety) and Greengenes (Lawrence Berkeley National Laboratory,Berkeley, Calif. (www.greengenes.lbl.gov); see, e.g., DeSantis et al.,“Greengenes, a Chimera-Checked 16S rRNA Gene Database and WorkbenchCompatible with ARB,” 72 Appl. Environ. Microbiol. 5069-72 (2006), whichis incorporated by reference herein in its entirety). The GenBank®genetic sequence database contains publicly available nucleotidesequences for almost 260,000 formally described species (NationalInstitutes of Health, Bethesda, Md. (www.ncbi.nlm.nih.gov); see, e.g.,Benson et al., “GenBank,” 41 Nucl. Acids Res. D36-42 (2013).

The sequence used for matching and identification may include the 16Sribosomal region, 18S ribosomal region or any other region that providesidentification information. The desired variant may be a genotype (e.g.,single nucleotide polymorphism (SNP) or other type of variant) or aspecies containing a specific gene sequence (e.g., a sequence coding foran enzyme or protein). An organism or species may also be identified bymatching its sequence to a custom internal sequence database. In somecases, one may conclude that a certain species or organism is found at alocation on the microarray if the sequence obtained from the portion ofthe sample at the location has at least a specified percentage identity(e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identity) to the known DNA, cDNA, or RNA sequence obtainedfrom that species or microorganism.

The disclosure further relates to a method of manufacturing a microarraycomprising a plurality of locations for applying a sample, wherein atleast one location is marked with a unique tag, the method comprisingthe steps of: (a) synthesizing a plurality of tags, wherein each tagcomprises a nucleic acid molecule comprising: (i) a location-specificnucleotide sequence; and (ii) a target-specific nucleotide sequence; and(b) placing a tag on at least one location of the plurality of locationson the microarray. In an alternative embodiment, the disclosure relatesto a method of manufacturing a microarray comprising a plurality oflocations for applying a sample, wherein at least one location is markedwith a unique tag, the method comprising the steps of: (a) synthesizinga plurality of tags, wherein each tag comprises a nucleic acid moleculecomprising: a target-specific nucleotide sequence and not comprising alocation-specific nucleotide sequence; and (b) placing a tag on at leastone location of the plurality of locations on the microarray. Thetarget-specific sequence may be the same at every location in themicroarray. In either of the above embodiments, step (a) may beperformed before step (b). The placing step (b) may comprise placing thetag at each location by a liquid handling procedure (for example,pipetting, spotting with a solid pin, spotting with a hollow pin, ordepositing with an inkjet device). At least one tag may include anucleic acid molecule or a portion of a nucleic acid molecule that ispre-synthesized. Step (a) may be performed simultaneously with step (b).In certain embodiments, at least one tag comprises a nucleic acidmolecule that is synthesized at each location by in situ synthesis. Thesynthesizing step (a) may comprise inkjet printing synthesis orphotolithography synthesis.

Each location on a microarray may be configured to receive a portion ofthe sample. A location may be tagged or labeled with a nucleic acidmolecule (e.g., an oligonucleotide) that comprises at least one of: (i)a location-specific nucleotide sequence (e.g., a barcode); and (ii) atarget-specific nucleotide sequence. A target-specific nucleotidesequence may complement or substantially complement a nucleotidesequence found in the sample. The order of the nucleotide sequences fromthe 5′ end to the 3′ end in the nucleic acid molecule may be: (1) alocation-specific nucleotide sequence; and (2) a target-specificnucleotide sequence. Alternatively, the order of the nucleotidesequences from the 5′ end to the 3′ end in the nucleic acid molecule maybe (2) then (1). The nucleic acid molecule may be attached at its 5′ endto the microarray. One or more locations on the apparatus (e.g.,microarray) may be untagged or unlabeled.

The terms “complementary” or “substantially complementary” may refer tothe hybridization, the base pairing, or the formation of a duplexbetween nucleotides or nucleic acids, such as, for instance, between thetwo strands of a double stranded DNA molecule or between anoligonucleotide primer and a primer binding site on a single strandednucleic acid. Complementary nucleotides are, generally, A and T/U, or Cand G. Two single-stranded RNA or DNA molecules are said to besubstantially complementary when the nucleotides of one strand,optimally aligned and compared and with appropriate nucleotideinsertions or deletions, pair with at least about 80% of the nucleotidesof the other strand, usually at least about 90% to 95%, and morepreferably from about 98 to 100%. Alternatively, substantialcomplementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically,selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, at leastabout 75%, or at least about 90% complementary.

The term “selectively hybridize” or “selective hybridization” may referto binding detectably and specifically. Polynucleotides,oligonucleotides and fragments thereof selectively hybridize to nucleicacid strands under hybridization and wash conditions that minimizeappreciable amounts of detectable binding to nonspecific nucleic acids.“High stringency” or “highly stringent” conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein. An example of “high stringency” or “highly stringent”conditions is a method of incubating a polynucleotide with anotherpolynucleotide, wherein one polynucleotide may be affixed to a solidsurface such as a membrane, in a hybridization buffer of 6×SSPE or SSC,50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS.

The nucleic acid molecule that is part of a location tag may comprise atleast one deoxyribonucleotide or at least one ribonucleotide. Thenucleic acid molecule may be single-stranded or double-stranded. Anucleic acid molecule may be a double-stranded molecule having asingle-stranded overhang.

In some embodiments, the location tag may be used to amplify a nucleicacid molecule that anneals to it. Thus, the location tag may comprise anucleic acid sequence that further comprises an amplification primerbinding site. An amplification primer binding site may be at least 16,at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, or at least 30 nucleotides in length. Theorder of the nucleotide sequences from the 5′ end to the 3′ end in thenucleic acid molecule may be, for example: (1) the amplification primerbinding site; (2) the location-specific nucleotide sequence; and (3) thetarget-specific nucleotide sequence.

In some embodiments, a nucleic acid molecule may comprise atarget-specific nucleotide sequence without comprising alocation-specific nucleotide sequence. In certain embodiments, a nucleicacid molecule may comprise a target-specific nucleotide sequence withoutcomprising either a location-specific nucleotide sequence or anamplification binding site sequence. In further embodiments, a nucleicacid molecule may comprise only a target-specific nucleotide sequence.In even further embodiments, a nucleic acid molecule may contain only atarget-specific nucleotide sequence. The amplification primer bindingsite may be capable of binding to a polymerase chain reaction primer, amultiplexed polymerase chain reaction primer, a nested polymerase chainreaction primer, a ligase chain reaction primer, a ligase detectionreaction primer, a strand displacement primer, a transcription basedprimer, a nucleic acid sequence-based primer, a rolling circle primer,or a hyper-branched rolling circle primer. Additional primers may beadded to the microarray during an amplification reaction. For example,both 5′ and 3′ primers may be needed for a PCR reaction. Target-specificnucleotide sequences may be amplified in the locations containing targetnucleic acid molecules and may be detected by, for example, qPCR, endpoint PCR, and/or dyes to detect amplified nucleic acid molecules.

It may be desirable to sequence a nucleic acid molecule that anneals toa location tag or the amplified product based on such a nucleic acidmolecule. The location tag may comprise a nucleic acid sequence thatfurther comprises an adapter nucleotide sequence. In certainembodiments, an adapter nucleotide sequence may not be found in thelocation tag but is added to the sample nucleic acid molecules in asecondary PCR reaction or by ligation. An adapter nucleotide sequencemay be a generic adapter or an adapter for a specific sequencingplatform (e.g., Illumina® or Ion Torrent™). An adapter nucleotidesequence may include a sequencing primer binding site. A sequencingprimer binding site may be capable of binding a primer for Sangersequencing, sequencing by hybridization, sequencing by ligation,quantitative incremental fluorescent nucleotide addition sequencing(QIFNAS), stepwise ligation and cleavage, fluorescence resonance energytransfer, molecular beacons, TaqMan reporter probe digestion,pyrosequencing, fluorescent in situ sequencing (FISSEQ), wobblesequencing, multiplex sequencing, polymerized colony (POLONY) sequencing(see, e.g., US 2012/0270740); nanogrid rolling circle (ROLONY)sequencing (see, e.g., US 2009/0018024), allele-specific oligo ligationassay sequencing, sequencing on an NGS platform, or any suitablesequencing procedure. Non-limiting examples of NGS platforms includesystems from Illumina® (San Diego, Calif.) (e.g., MiSeg™, NextSeg™,HiSeg™, and HiSeq X™), Life Technologies (Carlsbad, Calif.) (e.g., IonTorrent™), and Pacific Biosciences (Menlo Park, Calif.) (e.g., PacBio®RS II).

A location-specific nucleotide sequence (e.g., a barcode) may be atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, orat least 30 nucleotides in length.

A target-specific nucleotide sequence may be at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25 nucleotides, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 40, at least50, at least 75, or at least 100 nucleotides in length.

At least one location on a microarray may be further marked with aunique molecular identifier tag. Unique molecular identifiers may beused to quantify growth (e.g., growth of a microorganism colony orreplication of cells at the location). Unique molecular identifiers maybe random nucleotide sequences. Methods using unique molecularidentifiers and examples of unique molecular identifiers have beendescribed in the art, see, e.g., WO 2013/173394, which is incorporatedby reference herein in its entirety. For example, a unique molecularidentifier tag may have the nucleotide sequenceNNNANNNCNNNTNNNGNNNANNNCNNN (SEQ ID NO:1), wherein the Ns (equal randommix of ACGT) create a large encoding space so that each moleculeamplified gets a unique (specific) DNA sequence barcode (4̂N barcodes, or4̂21˜4 trillion in this example) This sequence can be counted withoutinterference from amplification bias or other technical problems. Thefixed bases in SEQ ID NO:1 (the A, C, G, T) help with reading thebarcode accurately, e.g., handling indels.

The present disclosure encompasses using location-specific tags tomonitor the presence or amount of more than one target-specificnucleotide sequence in a sample (e.g., multiplexing). At least onelocation on a microarray may be further marked with a second unique tagcomprising a nucleic acid molecule comprising, for example: (i) anamplification primer binding site; (ii) a location-specific nucleotidesequence; and (iii) a target-specific nucleotide sequence.

In some embodiments, a nucleic acid molecule may comprise atarget-specific nucleotide sequence without comprising alocation-specific nucleotide sequence. In certain embodiments, a nucleicacid molecule may comprise a target-specific nucleotide sequence withoutcomprising either a location-specific nucleotide sequence or anamplification binding site sequence. In further embodiments, a nucleicacid molecule may comprise only a target-specific nucleotide sequence.In even further embodiments, a nucleic acid molecule may contain only atarget-specific nucleotide sequence. The target-specific nucleotidesequence may be at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25 nucleotides, at least 26, at least 27, at least 28, at least29, at least 30, at least 40, at least 50, at least 75, or at least 100nucleotides in length. In certain embodiments, the target-specificsequence may be the same at every location in the microarray. Additionaltarget-specific nucleotide sequences may be monitored. For example, oneor more locations may be marked with at least 10, at least 25, at least50, at least 75, or at least 100 unique tags, wherein each tag comprisesa target-specific nucleotide sequence that is different from the othertarget-specific nucleotide sequences in the tags at that location.

Any genetic locus of interest may provide a target-specific nucleotidesequence. For example, sequences of bacterial 16S ribosomal RNA (rRNA),18S ribosomal RNA, poly(A) RNA, an RNA polymerase gene, a DNA polymerasegene, the RecA gene, a transposase gene, ribosomal internal transcribedspacer (ITS) sequences, a gene encoding an enzyme, control region DNAsequences, binding site DNA sequences, or a portion of any of thesesequences may serve as a target-specific nucleotide sequence. Adisclosed system, kit, apparatus, or method may use one or more of thebacterial 16S rRNA primers described in Sundquist et al., “BacterialFlora-Typing with Targeted, Chip-Based Pyrosequencing,” 7:108 BMCMicrobiology (2007) and Wang et al., “Conservative Fragments inBacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNAAmplicons in Metagenomic Studies,” 4:10 PLoS ONE e7401 (2009), each ofwhich is incorporated herein by reference in its entirety.

A sample used in the disclosed apparatuses and methods may comprise aplurality of nucleic acid molecules. A sample may comprise at least oneDNA molecule or at least one RNA molecule. A sample may comprise atleast one nucleic acid molecule formed by restriction enzyme digestion.A sample may comprise at least one cell (e.g., an archaebacterial cell,a eubacterial cell, a fungal cell, a plant cell, and/or an animal cell).A sample may comprise at least one microorganism. A sample may compriseone or more viruses (e.g., a bacteriophage), for which host cells mayneed to be provided. A portion of a sample at a location on a microarraymay be a single cell or a colony grown from a single cell. For example,individual microorganisms or cells may be placed in microwells and theindividual microorganisms or cells may be allowed to divide or replicateso that a colony grows within each microwell that had an individualmicroorganism or cell placed in it. A location on a microarray may thuscontain a single microorganism species or a mixed community ofmicroorganism strains that support one another's growth. A sample maycomprise any suitable dilutant. In non-limiting examples, a samplecomprises soil, sewage, fecal matter, contents of a body cavity, abiological fluid, living organic matter, dead organic matter, amicrobial suspension, naturally-sourced freshwater, drinking water,seawater, wastewater, supercritical carbon dioxide, a mineral, a gas, abuffer, alcohol, an organic solvent, and/or an oil. In some embodiments,a nucleic acid molecule comprising (a) (i) a location-specificnucleotide sequence and (ii) one or more target-specific nucleotidesequences; or (b) one or more target-specific nucleotide sequences(i.e., not comprising a location-specific nucleotide sequence) is placedon at least one location on a microarray before a portion of a sample isplaced at the location. In other embodiments, a nucleic acid moleculecomprising (a) (i) a location-specific nucleotide sequence and (ii) oneor more target-specific nucleotide sequences; or (b) one or moretarget-specific nucleotide sequences (i.e., not comprising alocation-specific nucleotide sequence) is placed on at least onelocation on a microarray after a portion of a sample is placed at thelocation. In one example, a sample or a portion of a sample may beplaced on a microarray and incubated before a nucleic acid moleculecomprising at least one of: (i) a location-specific nucleotide sequenceand (ii) a target-specific nucleotide sequence is placed on themicroarray. In some embodiments, a portion of the portion of a samplemay be removed from at least one location on the microarray and storedin a separate receptacle or the microarray may be split either before orafter the nucleic acid molecules are placed on at least one location onthe microarray.

At least one location-specific tag may comprise a nucleic acid moleculeor a portion of a nucleic acid molecule that is pre-synthesized andplaced at the location by a liquid handling procedure. For instance, aliquid handling procedure may be pipetting, spotting with a solid pin,spotting with a hollow pin, or depositing with an inkjet device. A tagmay be generated at the location using multiple nucleic acid moleculesthat are pre-synthesized separately. At least one tag may comprise anucleic acid molecule that is synthesized at the location by in situsynthesis (e.g., by inkjet printing or by photolithography).

Digital Enumeration of Species

A high density chip device comprised of a surface having high densitymicrowells is described herein. Microbes from a microbiome sample may bediluted and applied to the device such that wells contain approximatelyone microbe per occupied well. The chip then may be incubated such thatthe microbes replicate within the wells. Further, a DNA based locationalindexing system is described herein to determine what species is presentin each well. This indexing system may involve having PCR primerspreloaded into each well that contain addressing barcodes that identifythe well and a primer sequence targeted to a specific genetic element(e.g., 16S) in the microbial genome that provides species information ortargets a desired genetic sequence. After incubation, the microbial DNAis released, the PCR primers amplify the target bacterial DNA region,and the amplicons from the various wells on a chip are pooled and thenmay be read by next generation sequencing.

The systems, kits, apparatus, and methodologies described herein may beutilized to perform an absolute count of the number of each microbialspecies or variant in a sample. Each well may represent a digital eventwhich represents the presence of a single microbe in the originaldiluted sample. The locational indexing system may allow a user todetermine what bacterial species is in the well. A unit of measurementmay be “there is a bacterial species in a well” and may be independentof the number of bacteria in the well.

In one example, a mixed sample of microbes includes 50% Species 1, 30%Species 2, and 20% Species 3. The sample is diluted then applied to thechip such that each occupied well has, for the most part, one microbe.The microbe replicates. Note the replication rate may be different fordifferent species. Then, the chip is processed such that the DNA fromthe microbes in the wells is released and the 16S or some other targetsequence is amplified. The DNA amplification products from each well maybe pooled and sequenced using next generation sequencing. The nextgeneration sequencing data may be analyzed to determine, for each well,what species is in each occupied well. Many wells may not be occupied atall. The abundance of each species may be determined by: the totalnumber of wells occupied by each species divided by the total number ofoccupied wells. An absolute abundance determination may be made bymultiplying the % abundance of each species from step by the totalnumber of microbes in the original sample. The sequencing data may becompared to publicly available sequence datasets to determine whatspecies is in each occupied well. For example, ribosomal RNA sequencedata is available in the SILVA rRNA database project described above.Other ribosomal RNA sequence databases include the Ribosomal DatabaseProject, Greengenes, and the GenBank® genetic sequence database, alsodescribed above.

Current methods for estimating the abundance of microbial species in asample involve the use of traditional techniques such as microscopy,staining, selective media, metabolic/physiological screens, andcultivation using petri dishes. These methods are often inaccurate dueto lack of specificity (microscopy, staining, metabolic/physiologicalscreens) or lack of ability to account for all species in a sample(selective media, cultivation) whereby many species do not grow well ordo not grow at all with traditional approaches.

Current molecular methods for determining the relative abundance ofmicrobial species in microbiome samples involve extracting microbial DNAfrom samples, performing PCR amplification of the 16S or some other DNAregion that provide species or other information, then performing nextgeneration sequencing (NGS) on the resulting PCR product. The relativeabundance of each species in the original sample is inferred from therelative frequency of the species specific DNA sequence in the NGS data.There are many examples in the literature of this type of analysis andthis method underpins much microbiome research.

The problem with the current methodology is that it does not control fordifferent numbers of 16S gene that may exist in different microbes, PCRbias whereby sequences from different microbial species may be amplifiedat different rates, and sequencing bias where sequences from differentmicrobial species may be sequenced at different rates. The result isthat there is a lot of uncertainty with respect to the accuracy ofrelative abundance data derived using current methodologies.

The counting of different species may be based on the presence of aspecies in a single well. This is directly related to a single microbefrom the original sample partitioning into the well during loading. OnlyPCR/NGS may be used to identify what microbial species exists in eachwell. The number of sequences identified does not form part of thecalculation. Hence, it does not matter if there is PCR, NGS, or targetsequence copy number variance or bias in the method.

Some embodiments may have applications in microbiome research, microbialproduct discovery and development, clinical diagnostics, and any otherarea where accurate counts of microbial species in a sample arerequired.

Accordingly, some embodiments may provide a much more accuratemeasurement of the relative abundance of each species in a microbiomesample, and the ability to convert this relative abundance measurementinto an absolute abundance or a direct count of each species in theoriginal sample (by accounting for the dilution and/or combining with ameasurement of the total number of microbes in the original sample).Some embodiments may provide new applications for high densitymicrofabricated chips (in addition to cultivation and screening ofmicrobes).

FIG. 18 is a flowchart illustrating a counting method in accordance withsome embodiments. In step 1800, a sample is obtained. In step 1802, atleast one cell is extracted from the obtained sample. In step 1804, atleast one high density microwell array of a microfabricated device orchip is loaded with the at least one extracted cell. Step 1804 mayinclude preparing a cell concentration with the at least one extractedcell, selecting at least one nutrient/media, and/or selecting at leastone membrane. In step 1806, at least a portion of the microwell array issealed with the at least one selected membrane to retain the cellconcentration with the microwells. In step 1808, the chip is incubated.Step 1808 may include selecting a temperature, determining atmosphere(e.g., aerobic or anaerobic), and/or timing incubation). In step 1810,the cultivated cells may be sacrificed for identification. Step 1810 mayinclude PCR, sequencing, and/or various data analytics. In step 1812,information about the sample (e.g., a microbial community structure) maybe assessed and/or determined.

FIG. 19 is a diagram illustrating a counting method in accordance withsome embodiments. Panel 1900 shows examples of complex samples,specifically a microbiome sample 1902 and a soil sample 1904. In Panel1906, at least one cell is extracted from the sample using, for example,the protocol illustrated in FIGS. 5A and 5B. In Panel 1908, the at leastone extracted cell (and any environmental extract and/or dilutant) isloaded on a microfabricated device or chip with at least one highdensity microwell array 1910. Chip 1910 and a reagent cartridge 1912 maybe loaded into an incubator 1914. The reagent may be useful for addingliquid to maintain nutritional requirements for growth and/or variousscreening purposes. Panel 1916 shows the output: sequences and relativeabundance of cultivated cells.

Droplet-Based Platforms

A discrete droplet-based platform may be used to separate, cultivate,and/or screen in much the same way that chips are used. A droplet is ananalog of a microwell serving as a nano- or picoliter vessel. Dropletgeneration methods, especially when combined with cell-sorter-on-a-chiptype instrumentation, may be used to separate out microbes from acomplex environmental sample. Droplet addition may be used to feedmicrobes. Droplet splitting may be used for sequencing or some otherdestructive testing while leaving behind a living sample. All the prepwork necessary for sequencing may be done in droplet format as well.

Some embodiments may be used to get microbes out of a complexenvironment and into droplets. For example, a modular system forgenerating droplets containing cell suspensions may contain one or smallnumbers of cells. The aqueous drops may be suspended in a nonmiscibleliquid keeping them apart from each other and from touching orcontaminating any surfaces. Droplets may be generated at, for example,30 Hz in each microchannel, which translates into millions per day.

A drop-based microfluidic system may encapsulate, manipulate, and/orincubate small drops (e.g., about 30 pL). Cell survival andproliferation is noted to be similar to control experiments in bulksolution. Droplets may be produced at several hundred Hz, meaningmillions of drops can be produced in a few hours. A simple chip-baseddevice may be used to generate droplets and the droplets may beengineered to contain a single cell.

Some embodiments may be used to screen cells in droplets. Fluorescencescreening of droplets post-incubation may be done on-chip and at a rateof, for example, 500 drops per second. Droplets may be flowed through achannel at the focus of an epifluorescence microscope that may beconfigured for a number of different measurements. This may be aparticularly effective way to do screening for metabolites as the localconcentration is quite high on account of being confined to a very smalldroplet.

Some embodiments may be used to sort droplets. Once cells have beenisolated, grown, and/or screened, they may be sorted so that usefulsamples may be retrieved. Droplets may be sorted in an analogous way tothe commonly used FACS machine.

Some embodiments may be used to split droplets. Some embodiments mayrequire the ability to take a sample and split it in order to send oneportion to sequencing (a destructive process) and retain another portionthat is a viable culture. There are a number of different ways to splitdroplets including, but not limited to, constructing T-junctions withcarefully calculated dimensions that result in drops splitting as theyflow by or electrowetting (taking care not to cause cell lysis withvoltages that are too high).

Some embodiments may be used to merge droplets and/or add a reagent to adroplet. For example, long term incubation of cells (e.g., weeks)requires the ability to add liquid to maintain nutritional requirementsfor growth. It also may be useful to be able to add reagents for variousscreening purposes. Droplet screening relies on being able to merge adroplet containing a compound-code with a droplet containing a singlecell. The droplets then may be incubated and/or returned to an assaychip to identify compounds via their codes. This may require the abilityto precisely merge drops on an as-needed basis.

Some embodiments may be used to perform PCR in droplets. PCR may be usedin order to ultimately sequence a specific genetic element (e.g., the16S region) in order to identify microbes. This may be used to determinewhat type of microbe is growing in each well. In a droplet-based systemthis approach may be used to determine what microbe is present in eachdroplet as long as the correct primer sequence is designed to amplifythe right region of the genome.

Some embodiments may be used to sequence DNA out of droplets (e.g.,generated in the PCR step) and/or prepare DNA libraries.

Location Specific Tags for High Density Chips

A high density chip device having a surface with a high density ofmicrowells may be used. Microbes from a microbiome sample may be dilutedand applied to the device such that wells contain approximately onemicrobe per occupied well. The chip may be incubated such that themicrobe replicates within the well and the resulting populationrepresents a single species. A DNA-based locational indexing system maybe used to determine what species is present in each well. This indexingsystem may involve having PCR primers preloaded into each well thatcontain addressing barcodes that identify the well, and a primersequence targeted to a specific genetic element (e.g., 16S in themicrobial genome) that provides species information. After incubationthe microbial DNA may be released, the PCR primers may amplify thetarget bacterial DNA region, and the amplicons from the various wells ona chip may be pooled and then read by next generation sequencing.

The above locational indexing system may involve incorporating adifferent locational code for each well of the microchip, or multiplelocational codes may be incorporated into each well such that the totalnumber of codes required to code a specified number of wells is reduced.For example, if there are 100 wells in a chip it would require 100 codesif there is one code per well. The same chip could be coded with only 20codes if two codes were read from each well (i.e., 10 coding for the xaxis of the grid and 10 coding for the y axis).

An example of a PCR strategy to incorporate two codes per well isprovided in TABLE 2.

TABLE 2 Primers Amplified PCR Products 5′-CODE1-TARGETSEQUENCEPRIMER1-3′CODE1- 3′-TARGETSEQUENCEPRIMER2-CODE2- TARGETSEQUENCE- 5′ CODE 2

An example of a PCR strategy to incorporate three codes per wellprovided in TABLE 3.

TABLE 3 Primers Amplified PCR Products 5′-CODE1-TARGETSEQUENCEPRIMER1-3′CODE1- 3′-TARGETSEQUENCEPRIMER2-CODE2- TARGETSEQUENCE- ADAPTER 5′CODE2-ADAPTER- 3′-ADAPTER′-CODE3′ 5′ CODE3

Three oligo primers are used to make a single PCR product. Advantages tothis system using two oligos to put a multi-partite barcode on one endof the molecule may include, for example, reducing maximum length ofoligos needed or making extra-long barcodes.

This approach can be generalized to incorporate n barcodes per reaction.The approach can also have different implementations such that thebarcodes are on the same side of the target sequence region. NGSsequencing adaptors may be added, and the full sequence for thepopulation of barcoded PCR products may be read using next generationsequencing.

In another implementation, a fixed code may be added to indicate samplenumber or plate number and allow pooling of multiple samples/plates in arun for two barcodes as shown in TABLE 4.

TABLE 4 Primers Amplified PCR Products 5′-PLATEA-CODE1- PLATEA-CODE1-TARGETSEQUENCEPRIMER1-3′ TARGETSEQUENCE-3′-TARGETSEQUENCEPRIMER2-CODE2-5′ CODE 2

In another implementation, a fixed code may be added to indicate samplenumber or plate number and allow pooling of multiple samples/plates in arun for three barcodes as shown in TABLE 5.

TABLE 5 Primers Amplified PCR Products 5′-PLATEA-CODE1- PLATEA-CODE1-TARGETSEQUENCEPRIMER1-3′ TARGETSEQUENCE- 3′-TARGETSEQUENCEPRIMER2-CODE2-CODE 2-ADAPTER- ADAPTER 5′ CODE3 3′-ADAPTER′-CODE3′ 5′

Note that in all cases the position of the barcode in the sequenceconveys information hence the CODE1, CODE2, and CODE3 barcodes do notnecessarily have to be different from each other in a particular well.

Making oligos and printing chips using a single code coding system arehigh cost. For example, a 10,000 well chip requires 10,000 singlebarcodes and 10,000 separate printing cycles to place those barcodesinto the wells on the chip. If a two-code system is used, thenpotentially only 200 barcodes are required with only 200 printing cyclesto manufacture chips. This represents a significant saving in oligocost, printing time and printing capital investment.

The use of dual barcoded PCR primers, followed by amplification andsequencing analysis, to provide locational data on DNA or DNA containingmoieties randomly partitioned onto a microfabricated chip may have highutility and relatively low cost.

FIG. 20 is a diagram illustrating an indexing system in accordance withsome embodiments. Microwell chip 2000 has N rows and M columns, therebyproducingN×M unique indices. A location of a microwell in chip 2000 maybe considered to have the coordinates (N, M). Each column has a commonreverse primer sequence (e.g., R1, R2, R3, . . . RM), and each row has acommon forward primer sequence (e.g., F1, F2, F3, . . . FN). Forexample, a unique tag targeted to a specific genetic element in, forexample, 16S ribosomal ribonucleic acid (rRNA) may include forwardprimer sequence F515 and reverse primer sequence R806. Following PCR ofchip 2000, the presence of the targeted genetic element may be mappedback to a unique microwell of origin based on the presence of a forwardprimer sequence and a reverse primer sequence. For example, the presenceof F515 and R806 directs a user to the microwell with coordinates (515,806) in chip 2000.

Variability Reduction for PCR Amplification Product Across MicrowellsContaining Bacteria

A DNA-based locational indexing system may be used to determine whatspecies is present in each well. This indexing system may involve havingPCR primers preloaded into each well that contain addressing barcodesthat identify the well, and a primer sequence targeted to a specificgenetic element (e.g. 16S) in the microbial genome that provides speciesinformation. After incubation the microbial DNA may be released, the PCRprimers amplify the target bacterial DNA region, and the amplicons fromthe various wells on a chip are pooled and then read by next generationsequencing.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include limiting amount of PCR primer in thewell during manufacture of the chip such that for the majority ofpossible sample DNA concentrations the amount of PCR primer willlimiting in the DNA amplification reaction, hence the amount of PCRproduct produced will be less variable across wells.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include limiting the number of PCR cycles onthe chip to less than 3 cycles, or less than 5 cycles, or less than 10cycles or less than 15 cycles, or less than 20 cycles or less than 25cycles or less than 30 cycles such that the amount of PCR productproduced will be less variable across wells vs. a full cycle PCRamplification protocol.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include limiting the amount of nucleotides inthe reaction mix so that the number of PCR amplicons produced is a morerelated to the amount of nucleotides than the amount of DNA in theoriginal sample. Microwells with a large amount of target DNA willexhaust the nucleotides early in the cycling process while microwellswith a small amount of target DNA will exhaust the nucleotides later inthe cycling process, but produce around the same amount of amplificationproduct.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include limiting the amount of nutrientavailable to microbes growing in the wells such that cells willreplicate until the media is exhausted then stop replicating.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include placing a dye in each well thatidentifies PCR product such that the signal gets brighter as more PCRproduct is produced. The intensity of the dye during each PCR cycle maybe monitored, and a sample may be taken from the well once the desiredsignal intensity is observed.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include using mixtures of hybridization beadscovered with oligos complementary to each well-specific bar code toselectively hybridize amplified DNA from each well. Once the beads aresaturated unbound DNA may be washed away releasing bound DNA from thebeads. The amount of DNA from each well will then be normalized at thesaturation limit of the beads.

Some embodiments for limiting the variability in the amount of PCRproduct across wells may include incubating chips for a long period oftime such that the fast growing microbes rapidly fill wells and ceasereplicating, and the slower growing microbes gradually fill wells andcease growing once approximately the same number of cells are in thewells

In some embodiments, use of barcoded primers and next generationsequencing (NGS) within the context of the chip format and method may beused to identify which species is growing in which well on the highdensity microchip. When an approximately equal number of bacteria occupyeach microwell in the chip, the signal from each well in the NGS datamay be approximately the same.

For example, in a typical NGS run generating 12 million sequence reads,if 24 chips are sequenced in the run, each having 10,000 microwells, andthere is the same number of bacteria per well, there is on average 50reads per well.

However, different bacteria grow at different rates so it is likely thatsome wells have few bacteria and some wells will have many bacteria.This potentially skews the NGS run so much that the wells with fewbacteria are not detected in the NGS analysis.

Hence, in the example of a typical NGS run generating 12 millionsequence reads, 24 chips are sequenced per run, each having 10,000microwells, half of which have 100 times more bacteria in them than theother half. The probability that the bacteria in the slow growing wellsare detected is markedly reduced. In this case:

(10,000×24)/2×100=12,000,000  (1)

(10,000×24)/2=120,000  (2)

The low frequency wells are represented at 1% of total. So of 12,000,000reads in an NGS run 120,000 will be from the low frequency wells—i.e.average of 1 read per well.

To minimize the impact of this phenomenon novel methods need to bedeveloped to help equalize the amount of PCR product across wells sothat all wells are detected in the NGS run.

Silicon-Based Microwell Chips for Microbial Isolation, Growth,Screening, and Analysis

A microfabricated device or chip may be composed at least in part ofsilicon instead of or in addition to plastic, glass, and/or polymers toallow for electrical measurements on a well-by-well basis. For example,the walls of each well may be isolated to create microcapacitors. Inanother example, an FET in each well such that the gate surface isexposed to the contents of the well. Instead of a purely silicon-basedchip, thin metal layers may be generated on top of an existing chip byplating, vapor deposition, and/or arc/flame spraying. This may add morefunctionality to a chip, utilize alternate methods of manufacture whichmay be cheaper and/or cleaner, and/or allow miniaturization for handheldand/or portable devices.

Some embodiments may allow for monitoring growth by electricalmeasurement. Impedance monitoring may be applied to measuremicroorganism (e.g., bacterial) growth. For example, impedance across atube containing Escherichia coli (E. coli) is compared to cell countstherefrom in Ur et al., “Impedance Monitoring of Bacterial Activity,”8:1 J. Med. Microbiology 19-28 (1975), which is incorporated herein byreference in its entirety. Measurements may be taken on other types ofbacteria including Pseudomonas, Klebsiella, and Streptococcus todemonstrate the effect is general. Wells may be filled with differentmedia in order to test growth conditions across different formulations.

Some embodiments may allow for screening by electrical measurement.Electrical measurements may be made on a well-by-well basis allowing forscreening. One example would be pH. There are a number of different waysto get a pH-dependent response from the gate of a device in a wellincluding, but not limited to, ISFETs and pH-meters. An array of wellswith embedded pH sensors may determine, electrically, which wellscontain microbes that are producing acidic or basic metabolites. Asimple example is screening for the production of lactic acid fromlactose. Bacteria is diluted out into wells, grown, and then fedlactose. Wells that record a drop in pH contain microbes capable ofmetabolizing lactose into lactic acid.

Some embodiments may allow for electrical measurements of redox probes.Another way to leverage electrical measurements is to look at howbacteria in wells affect a known redox probe. Essentially, a system withwell-defined response may be measured in the presence of bacteria anddeviations from expected behavior may be attributed to the bacterialsamples. A typical redox probe is something like ferricyanide;[Fe(CN)6]3-/4-. The reduction of ferricyanide to ferrocyanide is verywell characterized such that small changes in behavior, particularlyaround electron transfer from the electrodes, are discoverable. Thissystem is “label free” as it detects without having to directly modifythe bacteria themselves.

Antibodies that can recognize microorganisms (e.g., E. coli) may beimmobilized on ITO electrodes. Electron transfer resistance may bemeasured from the electrodes to a ferricyanide containing solution. E.coli binding to the electrode surface increases the resistanceproportional to the concentration of E. coli on the surface. This is oneexample of a family of measurements that may be made to detect specifictypes of organisms or metabolites using redox probes.

Working in silicon (or at least metals or metallized plastics) providesadvantages including, but not limited to, less expensive production ofchips (e.g., by piggybacking on existing technologies); integrateddetection capability allowing small and/or portable versions; additionalmeasuring capabilities not present in other materials (e.g., LCR, CV,etc.); integration of newly discovered chip-based detection modalitiesinto existing devices; and the combination of electrical measurementsand sequencing. These advantages would benefit any customer usinginterferometric detection.

Releasable Barriers to Protect Well-Specific Chemistries on a Chip

FIGS. 21A-21E are diagrams illustrating a chip with well-specificchemistries in accordance with some embodiments. In FIG. 21A, amicrofabricated device or chip is shown with a plurality of microwells.In FIG. 21B, microwell-specific chemistries have been disposed in eachmicrowell of the chip. In FIG. 21C, a sealant has been applied over themicrowell-specific chemistries in each microwell of the chip, therebypreventing interaction of the chemistries with further additions to thewells. In FIG. 21D, samples are loaded, and experiments are performed onthe samples in the microwells. In FIG. 21E, a trigger (e.g., heat)releases the microwell-specific chemistries for interaction with samplesin the wells.

Microwell chips may be manufactured, be cleaned, and/or have surfacestreated. The specific chemistries may be prepared separately and thendeposited into wells by, for example, using a method and/or device thatallows a specific set of chemicals to be directed to a specific well (orwells). A sealant then may be applied to protect the various chemistriesfrom the environment and/or be removed/released/disbursed with somedefined, external trigger.

A microfabricated device or chip may be manufactured to a specificdesign, for example, cleaned and/or surface treated to improve wetting.PCR primers may be printed or pin-spotted into specific wells. The chipmay be allowed to dry, and then a wax layer may be deposited byevaporation from an ethanol solution. Optimal concentration may be about1% v/v. Molten wax may be applied directly or an aqueous or alcohol waxsolution may be sprayed. Alternatively, spin coating or vapor depositionmay be used. Various waxes may be used including, but not limited to,glyceryl stearate with and without polyethylene glycol, cetearylalcohol, 1-hexadecanol, glyceryl ester of stearic acid, ceteareth-20(CAS Registry No. 68439-49-6), and some commercial products including,but not limited to, Lotionpro™ 165 (available from Lotioncrafter®,Eastsound, Wash.) and Polawax™ (available from Croda, Inc., Edison,N.J.). The underlying chemistry later may be released by, for example,heating until the wax melts. For these compositions it will be between50° C. and 70° C. It is important to be low enough not to damage anychemical component or to boil our aqueous solutions.

The key concept is of well-specific chemistry that is walled off fromthe chip until the experimenter triggers release. This method may beused for barcoding in wells, but it also may be applied more broadly toa whole range of different problems. Different chemistries that may beuseful to seal on a chip include, but are not limited to, antibiotics,fluors, dyes, PCR primers, lysis-promoters, antibodies, and/or tests forvarious metabolites. While wax is a good way to seal things that canlater be released by heating, other materials may be used to seal andrelease upon exposure to light, sonication, and/or some other trigger.The advantage of this method over simply adding reagents to the chip isone of control on a well-by-well basis. A similar effect might beachieved by printing chemicals into wells after doing microbialexperiments, but this introduces problems with time (the print run maybe as long as a day) and the fact that it is impossible to expose everywell for the same amount of time if each well is filled individuallyafter the microbes are on the chip. With a release mechanism every wellcan be exposed at the same time. In one example, wax may be depositedonto chips by solvent casting.

Isolated Microwells for Simplicity and/or Controlling Relative Abundance

A high-density chip device may comprise a surface having high-densitymicrowells. Microbes from a microbiome sample, or other cell types, maybe diluted and applied to the device such that wells containapproximately one microbe or cell per occupied well. These microwellsmay be sealed with semi-permeable membranes that allow nutrients todiffuse into the microwells but prevent all or at least some of themicrobes or cells from moving out of the microwells.

A sample of microbes or cells may be prepared and then sealed into achip using an impermeable or only-gas-permeable membrane. No reservoirof liquid sits on top of the chip or membrane and hence only thenutrient in the well at the time of sealing is available to supportgrowth of the microbes or cells in the microwell. Two reasons for thisfeature include: (1) simplicity in construction and workflow as thedevice need not have semi-permeable membranes or reservoirs, nutrientsdo not have to be added, and there is less potential for contamination;and (2) a check on the relative abundance of fast-growers by limitingtheir access to nutrients. For a sample containing some fast-growers andsome slow-growers, the fast-growers will rapidly be resource-limited intheir respective microwells and stop or slow growth while theslow-growers continue to grow. This provides for slow growers to berepresented at a higher relative abundance in the population of microbesacross the chip, compared to the case where the fast growers do not havea limiting amount of nutrient. This becomes important for downstreamprocessing when sequencing everything on a chip. It also provides abetter detection limit for rare species as the rare species are notoutgrown by fast growing species to a point that limits the ability ofthe system to detect them.

Current methods attempt to get all species to grow whether they arefast- or slow-growing by nature. This has the inevitable result thatfast-growers dominate communities and only increase in relativeabundance with time. Many types of downstream analysis such assequencing or fluorescence screening cannot resolve every species in agiven population but only those above a certain limiting relativeabundance. If the goal is to preserve diversity and detect rare species,then the fast-growers need to be limited in some way.

For an example that demonstrates this idea, consider the simple case ofa sample containing two species: one doubles every day and the otherdoubles every week as shown in TABLE 6. If the slow-grower is rare tobegin with, at 5% relative abundance, it soon becomes very rare as bothspecies grow.

TABLE 6 Unlimited Day 0 Day 1 Day 2 Day 3 Day 7 Day 14 Fast grower 19 3876 152 2432 311296 Slow grower 1 1 1 1 2 3 Total 20 39 77 153 2434311299 Rel. ab. fast 0.950 0.974 0.987 0.993 0.999 1.000 Rel. ab. slow0.050 0.026 0.013 0.007 0.001 0.000

If the fast-growers are limited by competition for nutrition and/orphysical space to grow, then the relative abundance of the slow-growerswill start to increase after some time has elapsed as shown in TABLE 7.

TABLE 7 Limited Day 0 Day 1 Day 2 Day 3 Day 7 Day 14 Fast grower 19 3850 50 50 50 Slow grower 1 1 1 1 2 3 Total 20 39 51 51 52 53 Rel. ab.fast 0.950 0.974 0.980 0.980 0.962 0.943 Rel. ab. slow 0.050 0.026 0.0200.020 0.038 0.057

High Density Microfabricated Arrays for Biobanking Cells

Biobanks are designed to give researchers access to a large number ofsamples from a large population in order to drive certain types ofresearch, such as disease-related biomarker discovery. The current stateof the art in biobanking provides for samples to be stored in tubes orlow-density plate format such as a 96-well or 384-well plate. This workswhen the number of samples to be stored is relatively low in number andthe samples themselves are discrete, isolated populations. Currentapproaches to biobanking become very cumbersome when storing samples,such as microbiome samples, where the number of samples may be high andthe number of different species or variants in each sample may extendfrom hundreds to thousands or many millions per sample. Using currentmethods, a laborious isolation protocol must be implemented to separateout individual species or variants either prior to or subsequent to thestorage step in order to access a desired species or variant.

The systems, kits, apparatus, and methodologies described herein may beapplied to biobank cells, microbes, viruses, and other biologicalentities. A high-density chip device comprised of a surface having highdensity microwells may have thousands, hundreds of thousands, ormillions of microwells per chip. For example, microbes from a microbiomesample (or another biological entity such as a different type of cell ora virus) may be diluted and applied to the device such that wellscontain approximately one microbe per occupied well. The chip then maybe incubated such that the microbe replicates within the well and theresulting population represents a single species. A nucleic acid-basedlocational indexing system may be utilized to determine what species ispresent in each well. This indexing system may involve having PCRprimers preloaded into each well. The PCR primers may contain addressingbarcodes that identify the well, and a primer sequence targeted to aspecific genetic element (e.g., 16S) in the microbial genome thatprovides species information or targets a desired genetic sequence.After incubation the microbial DNA is released, and the PCR primersamplify the target bacterial DNA region. The amplicons from the variouswells on a chip may be pooled and then read by next generationsequencing.

Using high-density microfabricated chips for biobanking provides for amultitude of species or variants within each sample to be stored asseparate populations without the need to implement a laborious isolationprotocol either before or after storage. Using the DNA-based locationalindexing system or a custom assay enables a simpler, generic approach toidentifying genetic signatures or characteristics of the contents ofeach microwell to give information such as, for example, speciesinformation. Additionally, the chip devices provide for an extremelyspace effective method of storing cell isolates. For example, a singlemicroscope slide dimensioned chip with 100,000 wells occupiessubstantially less space that the corresponding traditional storageformats. The chip format also may be useful for properly archiving andcurating samples and/or for managing subject (e.g., patient) informationdatabases by having one chip contain many different samples from asingle subject.

In order to bank cells using this apparatus, cells may be disposedand/or positioned in microwells of the apparatus. The cells in themicrowells may be treated to ameliorate the impact of storage thenplaced in appropriate storage conditions. For example, cells may betreated with agents such as glycerol to ameliorate the impact offreezing. Then the chip may be placed in appropriate storage conditionssuch as a freezer. The cells may be dehydrated, lyophilized, and/orfreeze dried, and the chip may be placed in appropriate conditions tosafeguard the dried cells. Additional structures may be added to thechip to further enhance its utility as a storage device. For example, amembrane or another structural element may be placed on top of the chipto seal at least some of the wells prior to storage.

A chip may be loaded with cells such that a portion of microwells on thechip are occupied by approximately one cell each. The chip is incubatedto allow for replication of cells. The chip is duplicated, and eitherthe original chip or the duplicate chip is used to identify the cells orspecies or gene signatures present in each well of the chip (by, e.g.,using the locational indexing system described above). The chip istreated and/or stored in appropriate conditions. The replication stepand/or the identification step may take place after storage rather thanbefore storage.

One or more cells from each strain of a pre-existing set of isolatedstrains may be disposed into separate microwells on a chip. The positionof the microwell into which each strain or cell type was placed may berecorded, and the chip may be treated and/or stored in appropriateconditions.

A version of the chip may be created in which preservative chemistry issequestered underneath a wax barrier in each well. Isolates may beallowed to grow sealed up inside a chip and then preserved at a laterdate by heat-induced release of the preservatives before banking.

Such apparatus may be used to store/biobank mixed microbiome samplessuch as microbiome samples from soil, human gut, seawater, oral cavity,skin, etc. A chip may be used to store other types of biologicalentities such as fungi, archaebacteria, human cells (includingreproductive cells), animal cells, and viruses.

The DNA locational indexing system may be used across all biologicalentity types to generate information regarding the content of each well.An entire chip may be screened for desired activity using custom assayssuch as antibody- or substrate-based assays. For example, a chip withbanked populations of T cells may be screened for a particularimmunological activity.

In one illustrative example, human stool microbiome samples may becollected from study participants. The stool microbes from eachindividual may be biobanked on chips to maintain both a record of thatindividual's microbiome as well as a sample of the microbiome to use asa source of target microbes at a later date. In another example, mixedpopulations of T cells or other immunological cells may be sampled fromindividuals during clinical or research studies or as part of atherapeutic workflow (e.g., cell therapy using ex vivo treated cells).In yet another example, soil biomes may be stored in conjunction withseed banking.

High Resolution Picking

A high accuracy/precision picking apparatus or system may be designed toexecute various picking functions from and/or to microwells on amicrofabricated chip described above. A target substrate or chip may bea microscope slide format (approximately 25 mm×75 mm×1 mm) withinjection-molded features on one surface. Microwells may be arranged ina grid pattern with about 4-8 mm well-free edge around the edges of thechip. Well size and spacing may be determined based on pickercapability. Microwells may be square with a size from about 25 μm toabout 200 μm along each edge and spacing in from about 25 μm to about100 μm between well edges. Microwells may be circular or hexagonalinstead. Well depths may be from about 25 μm to about 100 μm. Forexample, a 75 mm×25 mm slide with a 7 mm edge, 100 μm square wells with100 micron edge-to-edge spacing will have about 16,775 microwells.

A high accuracy/precision picking system may be designed to executevarious picking functions from and/or to portions of a membranecorresponding to microwells on a microfabricated chip, as describedabove. A membrane may be a thin sheet that has previously been used toseal growing bacteria into microwells. When peeled off the chip themembrane may retain an imprint of the microwell array as well as asample of bacteria on its surface after separation from the chip. Thus,the peeled membrane may act as a replicate of the bacteria growing inthe chip.

A high resolution picker may receive data input from a user. The inputmay include at least one pair of chip microwell coordinates such thatthe picker picks from and/or to the at least one input pair ofcoordinates. A sterilization routine may be performed between cycles. Ahigh resolution picker may be capable of operating in an anaerobicchamber.

A high resolution picker system may receive a chip and align the pickerwith the chip, for example, using fiducial markers and/or referencewells. The picker may pick, for example, cells growing in microwells onthe chip into, for example, 96- or 384-well plates containing growthmedia. The picker may include a single picking pin or a plurality ofpicking pins.

A high resolution picker system may receive a membrane and align thepicker with the membrane, for example, using reference well marks on themembrane. The picker may pick, for example, cells from the membraneinto, for example, 96- or 384-well plates containing growth media. Thepicking pin(s) may have a different shape (e.g., mushroom-shaped) and/orsurface (e.g., texture) for picking from a membrane. The system also mayinclude one or more mechanisms (e.g., a floating pin and/or vacuum) tohold and/or flatten the membrane.

A high resolution picker system may enable chip replication via achip-to-chip transfer. The picker may receive and align a first chipbased on fiducial markers and/or reference wells. The picker also mayreceive and align a second chip based on fiducial markers and/orreference wells. The picker may transfer, for example, cells growing inmicrowells on the first chip to microwells on the second chip.

A high throughput system for automatically picking a target species of aplurality of species of at least one biological entity cultivated in amicrofabricated device may include a port for receiving themicrofabricated device. The microfabricated device defines a highdensity array of microwells. Each microwell of the high density array ofmicrowells is configured to isolate and cultivate at least one speciesof the at least one biological entity and includes at least one tag of aplurality of unique tags. Each tag of the plurality of unique tagsincludes a nucleic acid molecule, which includes a target-specificnucleotide sequence for annealing to the at least one biological entityand a location-specific nucleotide sequence correlating to at least onemicrowell of the high density array of microwells. The system alsoincludes a high-resolution picking apparatus with at least oneprotrusion for picking the at least one biological entity from at leastone microwell of the high density array of microwells. The systemfurther includes an input device for receiving an indication of at leastone target-specific nucleotide sequence and at least one processorcommunicatively coupled to the input device and the high-resolutionpicking apparatus. The at least one processor acquires the indication ofthe at least one target-specific nucleotide sequence from the inputdevice, compares the at least one target-specific nucleotide sequence tothe plurality of unique tags, determines at least one microwell of thehigh density array of microwells including the target species based onthe comparison, and controls the high-resolution picking apparatus topick the at least one biological entity from the at least one determinedmicrowell of the high density array of microwells.

A high throughput system is disclosed for automatically picking a targetspecies of a plurality of species of at least one biological entitycultivated in a microfabricated device. The microfabricated devicedefines a high density array of microwells, each microwell of the highdensity array of microwells being associated with at least one uniqueprimer of the plurality of unique primers. The system includes a portfor receiving a membrane removed from the microfabricated device, themembrane having sealed each microwell of the high density array ofmicrowells to retain the at least one biological entity in the highdensity array of microwells, such that portions of the at least onebiological entity corresponding to the high density array of microwellsremain attached to the membrane following removal of the membrane. Thesystem also includes a high-resolution picking apparatus including atleast one protrusion for picking the at least one biological entity fromat least one membrane location corresponding to at least one microwellof the high density array of microwells, an input device for receivingan indication of at least one target-specific nucleotide sequenceassociated with the target species, and at least one processorcommunicatively coupled to the input device and the high-resolutionpicking apparatus. The at least one processor acquires the indication ofthe at least one target-specific nucleotide sequence from the inputdevice, compares the at least one target-specific nucleotide sequence tothe plurality of unique tags, determines at least one membrane locationcorresponding to at least one microwell of the high density array ofmicrowells comprising the target species based on the comparison, andcontrols the high-resolution picking apparatus to pick the portions ofthe at least one biological entity from the at least one determinedmembrane location.

The above-described embodiments can be implemented in any of numerousways. For example, embodiments may be implemented using hardware,software or a combination thereof. When implemented in software, thesoftware code can be executed on any suitable processor or collection ofprocessors, whether provided in a single computer or distributed amongmultiple computers.

Further, it should be appreciated that a computer may be embodied in anyof a number of forms, such as a rack-mounted computer, a desktopcomputer, a laptop computer, or a tablet computer. Additionally, acomputer may be embedded in a device not generally regarded as acomputer but with suitable processing capabilities, including a PersonalDigital Assistant (PDA), a smart phone or any other suitable portable orfixed electronic device.

Also, a computer may have one or more input and output devices. Thesedevices can be used, among other things, to present a user interface.Examples of output devices that can be used to provide a user interfaceinclude printers or display screens for visual presentation of outputand speakers or other sound generating devices for audible presentationof output. Examples of input devices that can be used for a userinterface include keyboards, and pointing devices, such as mice, touchpads, and digitizing tablets. As another example, a computer may receiveinput information through speech recognition or in other audible format.

Such computers may be interconnected by one or more networks in anysuitable form, including a local area network or a wide area network,such as an enterprise network, and intelligent network (IN) or theInternet. Such networks may be based on any suitable technology and mayoperate according to any suitable protocol and may include wirelessnetworks, wired networks or fiber optic networks.

The various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Additionally, suchsoftware may be written using any of a number of suitable programminglanguages and/or programming or scripting tools, and also may becompiled as executable machine language code or intermediate code thatis executed on a framework or virtual machine.

Further Examples

In one example, a membrane is used to “split” a microfabricated chipdescribed herein to create isolates from a two-component mixture. Freshcultures were started the night before. Prior to loading on a chip, thebacteria were counted (MACSQuant® Analyzer 10, Miltenyi Biotec),diluted, and mixed to produce a master solution of equal partsAcinetobacter Calcoaceticus (AC) and Saccharomyces Cerevisiae (SC).These two components were chosen because they are easily distinguishedin a typical inverted microscope.

The AC/SC mixture was diluted to target one cell isolated in eachmicrowell (for each microwell containing a microbe). The diluted AC/SCmixture was then loaded onto the microwells on the chip by pipetting themixture (or solution, suspension) onto the array of microwells on thesurface of the chip. As this is a random loading process, somemicrowells were loaded with one cell, some wells were empty, and somewith more than one cell per microwell. After loading the microwells, thearray was sealed with a polycarbonate membrane backed by PCR tape.Strong pressure was applied to be make sure the microwells were keptisolated from each other. The chip was then incubated overnight at 30 C.On the following day, the chip was checked by an inverted microscope forevidence of growth and of good sealing of the microwells. Images of thechip were stitched together using common stitching routines in Fiji (anopen-source image processing package). FIG. 24A shows a composite imagewhich was stitched together from a series of images taken from theinverted microscope of the chip (with the reservoir and membrane inplace). FIG. 24B is a close-up image taken from the inverted microscopeshowing some microwells with growth and some microwells empty. FIG. 25shows microscopy images of individual microwells as inspected for purecultures (SC, AC, and mixture thereof).

After some period of growth on a chip, the media reservoir was removedand the membrane was carefully peeled back to split the chip. The peeledmembrane was mounted to a backing material for ease of handing, andstored until ready for picking.

The chip was inspected for microwells that contained pure culture. Eightwells, four of each type, were identified and their respective location(X and Y coordinates) on the membrane determined. Each microwell left adistinct impression on the membrane that allowed for the counting of themicrowells (for example three across and two down from the upper rightcorner) as well as localizing the portion on the membrane that coveredand sealed that particular well.

Using a tiny pin mounted on a 3-axis controller on the deck of aninverted microscope, the membrane was first touched at selectedlocations and then dipped the pin into a tube of media to create purecultures. In between picks, the pin was sterilized with alcohol.

The eight small tubes were incubated at 30° C. for 48 hours and thenexamined. No contamination was observed. Three of four AC picks and oneof four SC picks were successful. FIGS. 26A and 26B show images of purecultures of SC (in FIG. 26A) and pure cultures of AC (in FIG. 26B).

In an example, the microfabricated chips herein described were used forhigh-throughput screening. Two strains of e. coli, one capable ofmetabolizing lactose to lactic acid (lac+) and one not (lac−) weremixed, diluted, and spread onto a chip. The lac+ bacteria were presentat 1% abundance and the total sample was diluted down to provide forapproximately one cell per occupied well. The bacteria were sealed undera polycarbonate membrane and then a silicone gasket was affixed to thearray. Warm Eosin methylene blue (EMB) agar was poured into the gasketand allowed to harden above the array. EMB agar is selective for certaintypes of Gram negative bacteria, and provides a colorimetric indicationfor lactose fermentation. Bacteria that ferment lactose into lactic aciddrops the local pH increasing dye absorption turning those colonies darkpurple or black. Non-fermenters do not drop the pH and may even increaseit. Those colonies remain clear. The chip was left to grow for 48 hours.It was then photographed in a microscope. Fourteen images were combinedto show a large number of 50-micron wells on the array.

The image shown in FIG. 22A contains 1800 microwells with 9 of themturning dark, indicating the presence of lac+ bacteria. The image shownin FIG. 22B is an enlarged portion of the image shown in FIG. 22A,showing the appearance of a dark microwell.

In a different experiment, the “hits” from a chip were recovered. EMBagar over a membrane was used to assay for dark wells, then a pin wasused to pick through the agar layer and membrane and transfer materialfrom the microwell to a small tube and start a new colony.

In some of the below examples, PCR was performed on a chip of thepresent disclosure using purified bacterial gDNA as sample. In thisprocess, PCR mastermix containing 16S rRNA V4 primers (for oligo printedchips, primers were not added to the mastermix) and template gDNA waspipetted onto the surface of the chip. A reservoir was placed over themicrowells on the chip and mineral oil added to avoid PCR bufferevaporation. Thermo cycling program used was 96° C. 10 min, 39 cycles of60° C. 2 min, 98° C. 40 second, 60° C. 2 min, then 10° C. for hold.After PCR, the chip was taken out, the oil removed, and amplicon washedoff the chip and the 16S rRNA V4 amplicon extracted with Qiagen Qiaquickkit. These amplicons were then amplified with sample index based NGSprimers by tube PCR to prepare NGS library. The tube PCR program is 95°C. 2 min, 35 cycles for 95° C. 35 seconds, 50° C. 45 seconds, 72° C. 45seconds. Then 72° C. min 2 min and 4° C. for hold. Gel electrophoresiswas used to determine presence of amplicon.

In some of the below examples, PCR was performed on a chip of thepresent disclosure using bacterial cells as sample. In this process,bacteria were loaded onto a chip by pipetting a bacterial suspensiononto the surface of the chip on which the microwells were located. Amembrane was applied to the chip to seal the bacteria in the wells andincubated the chips. After incubation at 30-37° C. for 1-5 days(incubation time and temperature dependent on the microbial speciesused), the bacteria in the microwells were centrifuged, the membraneremoved, and the medium absorbed out by blotting the chip lightly withabsorbent paper. PCR mastermix containing 16S rRNA V4 primers (for oligoprinted chips, primers were not added to the mastermix) was pipettedonto the surface of the chip. A reservoir was placed over the microwellson the chip and mineral oil added to avoid PCR buffer evaporation.Thermo cycling program used was 96° C. 10 min, 39 cycles for 60° C. 2min, 98° C. 40 seconds, then 60° C. 2 min and 10° C. for hold. AfterPCR, the chip was taken out, the amplicon washed off the chip and the16S rRNA V4 amplicon extracted with Qiagen Qiaquick kit. These ampliconswere then amplified with sample index based NGS primers by tube PCR toprepare NGS library. The tube PCR program was 95° C. 2 min, 35 cyclesfor 95° C. 35 seconds, 50° C. 45 seconds, 72° C. 45 seconds, then 72° C.10 min, and 4° C. for hold. Gel electrophoresis was used to determinepresence of amplicon.

An experiment was performed with E. coli gDNA as target. E. coli gDNAand primers and DNA polymerase were added in a PCR buffer for twodifferent microwell sizes—100 μm and 400 μm. The primers are designed toamplify the 16S region of bacterial DNA. The negative control is thesame process but without DNA polymerase in the reaction buffer. Aftergoing through chip PCR, Qiagen Qiaquick kit purification and a secondround of PCR in a tube, the result indicates that there is a bandcorresponding to 16S V4 amplicon (T1-1, T1-2), but not the negativecontrol (T2-1, T2-2). This confirms that the 16S rRNA V4 fragment didamplify on the chip. The 100 um wells appear to have a betteramplification (strong band). This data confirmed on-chip amplificationof 16S region from E. coli gDNA.

In a further test, genomic DNA from Microbial Mock Community B (20bacteria, BEI Resources HM-782D, Table 8) was used as the target samplefor the on-chip PCR. After chip PCR and NGS library amplification withsample index (FIG. 27, which shows gel electrophoresis for NGS library,lane 1-2 show the amplicon for 16S rDNA V4. Lane 1, amplicon from mockcommunity gDNA; lane 2, amplicon from mock community gDNA; lane 3,negative control; lane 4, DNA ladder), the NGS was run on the IlluminaMiSeq.

TABLE 8 Microbial Mock Community B NCBI Reference Organism SequenceAcinetobacter baumannii, NC_009085 strain 5377 Actinomycesodontolyticus, NZ_AAYI02000000 strain 1A.21 Bacillus cereus, strain NRSNC_003909 248 Bacteroides vulgatus, strain NC_009614 ATCC ® 8482 ™Cibstridium beijerinckii, strain NC_009617 NCIMB 8052 Deinococcusradiodurans, NC_001263, NC_001264 strain R1 (smooth) Enterococcusfaecalis, strain NC_17316 OG1RF Escherichia coli, strain K12, NC_000913substrain MG1655 Helicobacter pylori, strain NC_000915 26695Lactobacillus gasseri, strain NC_008530 63 AM Listeria monocytogenes,NC_003210 strain EGDe Neisseria meningitidis, strain NC_003112 MC58Propionibacterium acnes, NC_006085 strain KPA171202 Pseudomonasaeruginosa, NC_002516 strain PAO1-LAC Rhodobacter sphaeroides,NC_007493, NC_007494 strain ATH 2.4.1 Staphylococcus aureus, strainNC_010079 TCH1516 Staphylococcus epidermidis, NC_004461 FDA strain PCI1200 Streptococcus agalactiae, NC_004116 strain 2603 V/R Streptococcusmutans, strain NC_004350 UA159 Streptococcus pneumoniae, NC_003028strain TIGR4

The NGS data has total reads about 2550596 and pass quality filtering93%. From Kingdom to Genus, there are total reads classified totaxonomic level 72-73% and Species 57% (See FIG. 28). The NGS data showthat 95% of the genera for this mock community could be identified,except Propionibacterium acnes. 17 species out of total 20 (85%) wereidentified at the species level by 16S sequence. Table 9 shows that 17species from the gDNA mock community were identified at the specieslevel by the 16S NGS analysis. An additional two species (Listeria andLactobacillus) were identified at the genus level. The call for genusClostridium without species identification may be related withClostridium beijerinckii.

TABLE 9 Chip PCR NGS Data Analysis for Mock Community's Genus andSpecies Classification Kingdom Phylum Class Order Family Genus Speciesnum_hits %_hits Bacteria Thermi Deinococci Deinococcales DeinococcaceaeDeinococcus radiodurans 186727 14.158 Bacteria Firmicutes BacilliBacillales Listeriaceae Listeria innocua 80765 6.128 BacteriaProteobacteria Epsilonproteobacteria Campylobacterales HelicobacteraceaeHelicobacter pylori 51573 3.913 Bacteria Firmicutes BacilliLactobacillales Streptococcaceae Streptococcus mutans 42768 3.245Bacteria Proteobacteria Betaproteobacteria Neisseriales NeisseriaceaeNeisseria meningitidis 34539 2.621 Bacteria Firmicutes BacilliLactobacillales Streptococcaceae Streptococcus agalactiae 30704 2.33Bacteria Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium23726 1.8 Bacteria Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus taiwanensis 23293 1.767 Bacteria Bacteroidetes BacteroidiaBacteroidales Bacteroidaceae Bacteroides vulgatus 23258 1.765 BacteriaProteobacteria Gammaproteobacteria Pseudomonadales MoraxellaceaeAcinetobacter baumannii 16792 1.274 Bacteria Firmicutes BacilliBacillales Staphylococcaceae Staphylococcus aureus 12022 0.912 BacteriaProteobacteria Alphaproteobacteria Rhodobacterales RhodobacteraceaeRhodobacter sphaeroides 9574 0.726 Bacteria ProteobacteriaGammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonasaeruginosa 7155 0.543 Bacteria Proteobacteria GammaproteobacteriaEnterobacteriales Enterobacteriaceae Escherichia coli 6849 0.52 BacteriaActinobacteria Actinobacteria Actinomycetales ActinomycetaceaeActinomyces odontolyticus 3558 0.27 Bacteria Firmicutes BacilliBacillales Staphylococcaceae Staphylococcus epidermidis 1733 0.131Bacteria Firmicutes Bacilli Lactobacillales StreptococcaceaeStreptococcus pneumoniae 656 0.05 Bacteria Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus gasseri 298 0.023Bacteria Firmicutes Bacilli Bacillales Paenibacillaceae Paenibacillusprosopidis 233 0.018 Bacteria Firmicutes Clostridia ClostridialesClostridiaceae Clostridium beijerinckii 179 0.014

In another test, chip PCR with mock community bacteria cells wereperformed for NGS. 7 bacteria species were grown as internal mockcommunity, which included: Pseudomonas aeruginosa, Salmonellatyphimurium, Staphylococcus warneri, Serratia marcences, Bacilluscereus, Bacillus subtilis and E. coli. Same general procure describedabove was followed for chip PCR. The mock community mixture was loadedonto a chip, sealed with a membrane, incubated overnight, centrifugedthe chip for 3 min at 3000 rpm. The membrane was removed, then themedium absorbed with kimwipes. The chip was filled with PCR buffer and16S rRNA PCR primers, and thermocycling was performed according to themethod above. NGS DNA library was prepared using the procedure asdescribed above and sequenced with Illumina Miseq. FIG. 29 indicates aweak amplicon band from the amplified samples. Analysis of resulting NGSdata identified 6 genera from the internal mock community. Three speciesfrom the mock community were identified at the species level.

In a further example, a barcode (“locational tag”) system was includedfor printing into the microwells of a chip. An i5 index and i7 indexwere designed to separately link to the 16S rRNA V4 probe forward primerand reverse primer (see FIG. 30, where the index i5 and i7 are used forchip-well location and index i7′ is used for sample index for multiplexsamples assay. Each index was between 8-12 base pairs in length and wedesigned different combinations in order to identify each well on a2,500 microwell chip. A commercially available precision printinginstrument was used to print two oligos (each oligo containing one indexsequence and a 16S primer sequence) into each microwell (microwell size100 μm×100 μm×100 μm). Each microwell contained a different combinationof indexes, so therefore the indexing system provided for identificationof which PCR product derived from each microwell. After deposition ofthe PCR primers a wax layer was added to each microwell using a printingdevice and the method described elsewhere in the specification.Additionally, a sample index system was designed to allow labeling ofeach sample during NGS library preparation. Each sample index had 8-12base pair. The sample index was added during the second step PCR.Therefore, a single NGS run can generate sequence data from DNA derivedfrom multiple chip experiments and linked to chip on which the DNA wasgenerated. This can save the time and reduce NGS cost per sample.

Indexed oligos and wax were printed into microwells of a chip asdescribed above. PCR mastermix (without primers) and mock community gDNAare loaded into the chip for running a chip PCR. After chip PCR, asample index and NGS sequencing adapters were added for each sample ofchip amplicon in a second round of PCR. Four chips were run and fourdifferent sample index were applied. NGS was performed on all foursamples in a single run using a Miseq (Illumina, San Diego). Theseindexes were decoded using a computer program and NGS data from thesefour samples were extracted. Sample data shows that microwell specificindexes across the chip were successfully read by NGS. 37% of wells havemore than 300 sequence reads (>×300 sequence, 925 wells), 25% wells have100-299 sequence call, 28% wells have 1-99 sequence call and only 10%wells have 0 sequence call (see FIG. 31). Such results demonstratedprinted chip with dual index oligo workflow.

In one aspect, a method of screening for at least one biologicalinterest of interest in a sample is provided. The method employs amicrofabricated device as described herein that has a top surfacedefining an array of microwells. At least one microwell of the array ofmicrowells is loaded with: (a) at least one cell from a sample; (b) anindicator substance; and (c) an amount of a nutrient. The cell, theindicator substance, and the nutrient can be loaded sequentially, in anyorder, or any of their combinations can be loaded sequentially orsimultaneously. A membrane is applied to the microfabricated device toretain the at least one cell in the at least one microwell. The membranecan be a membrane that is permeable only by gas (e.g., oxygen), or amembrane that is liquid permeable, or impermeable. Components of thenutrient may permeate through the membrane from inside of the microwellto the outside, or a reservoir of nutrient can be provided initiallyoutside the membrane and exterior to the microwell, and the nutrient canpermeate through the membrane into the microwell. The microfabricateddevice is incubated at predetermined conditions (e.g., at desiredtemperature/atmosphere and other conditions as appropriate for the cell)for a duration of time to grow a plurality of cells from the at leastone cell in the at least one microwell. An optical property, e.g.,color, optical density, fluorescence, phosphorescence, or luminescenceof the at least one microwell can be evaluated. The optical property canbe evaluated as a collective or average property for each individualmicrowell, or can be evaluated in more details in each of the wells,e.g., as optical property patterns such as optical morphology. Thisevaluation can be done at the completion of the incubation, and/or atmultiple points in time, e.g., after loading the microwell contents butbefore incubation, and after the incubation has begun but has notfinished, etc. Based on the optical property evaluated or the comparisonacross multiple times of evaluation of the optical property, a presenceor absence of at least one biological entity of interest in the at leastone microwell can be determined.

As used herein, the “indicator substance” refers to a chemical thatrenders a microwell in which it is located with certain distinct opticalproperty before incubation, after the incubation is complete, and/or ata point during incubation but before the incubation has been completed.Such optical property may change due to a reaction or interaction of theindicator substance with a cell (e.g., migration into a cell, ingestedby a cell, etc.), a cell component (e.g., binding to a cell membrane,binding to a DNA of a cell, etc.), or a substance produced by a cell ina subject microwell. The change in optical property may be due to achange in the amount of the indicator substance (e.g., part or all of ithas been converted to a different compound due to a reaction with a cellcomponent or cell product), or a reorganization of the structure of theindicator substance, or the binding of the indicator substance toanother component.

In some embodiments, the indicator substance can be an inorganicphosphate compound, e.g., calcium phosphate, which is insoluble inwater. When deposited in a microwell and observed by a microscope,calcium phosphate particulates can be observed. This compound can beused to screen phosphate solubilizing bacteria, which can producecertain acids to solubilize the compound. As a result, the phosphate candiminish or disappear. Other inorganic insoluble phosphates such asaluminum phosphate could also be used. Comparisons of the microwell canbe performed, e.g., by capturing digital images of the microwell undermicroscope and analyzing the change in color, optical density,morphology, or other characteristics of the images by computer software.

In an example of the embodiments, calcium phosphate was deposited inmicrowells of a microfabricated device using an accurate printing deviceby printing specific volumes and concentrations of a source of calcium(e.g., calcium chloride) and a source of phosphate (e.g. sodiumphosphate). In this manner, the starting materials were easily dispensedliquids, which then reacted to form solid calcium phosphate at thebottom of each microwell. The result was a microfabricated device with acontrolled amount of calcium phosphate at the bottom of each well.Bacterial strains, one positive for phosphate solubilizing capabilityand one negative for phosphate solubilizing capability, were dilutedsuch that microwells contain individual bacteria and some nutrients. Amembrane was applied on the top surface of the microfabricated deviceusing pressure and heat, sealing the contents of the microwells. Themicrofabricated device was incubated overnight to grow more cells. Themicrofabricated device was observed under an optical microscope atvarious points in time. Microwells containing a strain capable ofsolubilizing phosphate was easily identified because the calciumphosphate at the bottom of the well disappeared when it was solubilized.As shown in FIGS. 32A-32D, under high magnification individual wellsshowed phosphate unchanged because there were no cells present (in FIG.32A), phosphate solubilizing cells had grown and solubilized phosphate(in FIG. 32B), non-phosphate solubilizing cells have grown but thephosphate did not change (in FIGS. 32C, 32D).

The location of each microwell where the calcium phosphate haddisappeared could be provided to a picking device, which can be used totransfer at least a portion of the contents of the microwell to a targetlocation, e.g., a 96-well plate for further incubation/growth. Aftergrowth in the 96-well plate, the amount of material can be sufficientfor a variety of further testing and identification procedures.

In some embodiments, the indicator substance can be a pH sensitive dye.For example, the pH sensitive dye can change color in a visible lightrange within a selected range of pH. The pH sensitive dye can also befluorescent in a first range of pH and not fluorescent in a second,different range of pH. It can also change its fluorescencecharacteristics, e.g., wavelengths for absorbing incident light,transfer efficiency, reemission wavelengths, etc. when pH is changed.The pH sensitive dye can be deposited separately into the microwells, orincluded in the media or nutrient in which microbes are grown. If duringthe growth and/or proliferation of the cells in the microwells an acidor base is produced, the color of this dye may change.

In examples of the embodiments, pH-sensitive dyes such as chlorophenolred, bromocresol purple, and bromothymol blue, were used as theindicator substances. In one example, bromocresol purple, which changesfrom purple at pH 7 (and above) to yellow at pH 5 (and below), was used.It was included in the media or nutrient in which microbes were grown.Bacterial strains were loaded into microwells of a microfabricateddevice with nutrient, sealed with a membrane, and allowed to growovernight. The microfabricated device was observed under an opticalmicroscope at various points in time. From the observed color change ina microwell (either by naked eye or by image analysis by computersoftware), those microwells that contained a strain that produces a pHinfluencing chemical was identified.

In some embodiments, pH sensitive chemiluminescent dyes, such asluminol, can be used as the indicator substance. In further embodiments,pH sensitive photoluminescent (fluorescent or phosphorescent) dyes ormaterials can be used as the indicator substance (or part of theindicator substance). For example, a pH sensitive fluorescent dye can befluorescent in a first range of pH and not fluorescent in a second,different range of pH. For another example, a fluorescent dye can havedifferent fluorescence characteristics at different pH. In the case of aphosphorescent material, a phosphor can be coated with many different pHsensitive dyes. Due to energy transfer from excited phosphor to thecoated dyes, the dye that is most absorbing at a given local pH wouldmake emits the brightest.

In some embodiments, the indicator substance can include a redoxindicator whose optical property can depend on a specific electrodepotential. For example, resazurin (7-Hydroxy-3H-phenoxazin-3-one10-oxide), which is a blue dye and weakly fluorescent, can be reduced tothe pink colored and highly red fluorescent resorufin. Resazurin can beused as an indicator substance to determine if a microwell contains livebacteria.

A microfabricated device may include a substrate with a series offunctional layers. The series of functional layers includes a firstfunctional layer defining a first array of experimental units (e.g.,wells) and at least one subsequent functional layer defining asubsequent array of experimental units (e.g., microwells) in eachexperimental unit of the preceding functional layer. Each of theexperimental units may be configured to receive and grow at least onecell, perform at least one screen, and/or test at least one nutrient.

In an embodiment, an apparatus for screening different conditionsagainst a matrix of cells includes a substrate. The substrate includes afirst surface. The surface defines a first array of wells. Each wellincludes an inner surface. Each inner surface defines a second array ofmicrowells. Each microwell is configured to receive and grow at leastone cell.

In an embodiment, an apparatus for screening different conditionsagainst a matrix of cells includes a substrate with a series offunctional layers. The series of functional layers includes a firstfunctional layer defining a first array of experimental units and atleast one subsequent functional layer defining a subsequent array ofexperimental units in each experimental unit of the preceding functionallayer. Each of the experimental units is configured to receive and growat least one cell.

In an embodiment, a method is disclosed for segregating cells in asubstrate including a first surface. The first surface defines a firstarray of experimental units. Each experimental unit is configured toreceive at least one cell. At least a portion of each experimental unitis configured with first surface characteristics that include attractingcells and/or increasing cellular tendency to occupy the experimentalunit. Alternatively or in addition, at least a portion of the firstsurface may be configured with second surface characteristics thatinclude repelling cells and/or reducing cellular tendency to stick tothe first surface. The method also includes applying a compositionincluding cells to the first surface such that at least one celloccupies at least one experimental unit.

In an embodiment, a method for sampling a cell population in a substrateincludes sampling a population of cells in at least one microwell byapplying a picking device to a first surface of the substrate. Thedevice includes at least one protrusion facing the first surface. The atleast one protrusion has a diameter less than the opening diameter ofeach microwell. The at least one protrusion is inserted into at leastone microwell holding a population of cells such that a portion of thepopulation of cells in the at least one microwell adheres and/orattaches to the at least one protrusion. The method also includeswithdrawing a sample of the population of cells in the at least onemicrowell by removing the device from the first surface of the substratesuch that the portion of the population of cells in the at least onemicrowell remains adhered and/or attached to the at least oneprotrusion.

In an embodiment, a method for sampling a cell population in a substrateincludes sampling a population of cells in at least one experimentalunit by applying a picking device to a first surface of the substrate.The device includes at least one protrusion facing the first surface.The at least one protrusion has a diameter less than the openingdiameter of each microwell. The at least one protrusion is inserted intoat least one experimental unit holding a population of cells such that aportion of the population of cells in the at least one experimental unitadheres and/or attaches to the at least one protrusion. The method alsoincludes withdrawing a sample of the population of cells in the at leastone experimental unit by removing the device from the first surface ofthe substrate such that the portion of the population of cells in the atleast one experimental unit remains adhered and/or attached to the atleast one protrusion.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface and a second surface opposite the firstsurface, the first surface defining a first array of microwells,includes applying a picking device to the second surface of thesubstrate. The device includes at least one protrusion facing the secondsurface. The at least one protrusion has a diameter about equal to orless than the diameter of each microwell. The at least one protrusion ispushed against the second surface at a location corresponding to atleast one microwell holding a population of cells and/or inserted intothe at least one microwell holding the population of cells such that aportion of the population of cells in the at least one microwell isdisplaced above the inner surface of the well and/or the first surfaceof the substrate. The method also includes sampling the population ofcells in the at least one microwell by collecting the displaced portionof the population of cells.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface and a second surface opposite the firstsurface, the first surface defining a first array of experimental units,includes applying a picking device to the second surface of thesubstrate. The device includes at least one protrusion facing the secondsurface. The at least one protrusion has a diameter about equal to orless than a diameter of at least one experimental unit. The at least oneprotrusion is pushed against the second surface at a locationcorresponding to the at least one experimental unit holding a populationof cells and/or inserted into the at least one experimental unit holdingthe population of cells such that a portion of the population of cellsin the at least one experimental unit is displaced above the firstsurface of the substrate. The method also includes collecting thedisplaced portion of the population of cells.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface, the first surface defining a first array ofwells, each well having an inner surface defining a second array ofmicrowells, each microwell having an opening diameter, includes applyinga picking device to the first surface of the microfabricated substrate.The device includes at least one protrusion facing the first surface.The at least one protrusion has a diameter less than the diameter ofeach microwell. The at least one protrusion is inserted into at leastone microwell holding a population of cells such that a portion of thepopulation of cells in the at least one microwell is volume displacedabove at least one of the inner surface of the well and the firstsurface of the substrate. The method also includes sampling thepopulation of cells in the at least one microwell by collecting thevolume displaced portion of the population of cells.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface, the first surface defining a first array ofexperimental units, includes applying a picking device to the firstsurface of the microfabricated substrate. The device includes at leastone protrusion facing the first surface. The at least one protrusion hasa diameter less than a diameter of at least one experimental unit. Theat least one protrusion is inserted into the at least one experimentalunit holding a population of cells such that a portion of the populationof cells in the at least one experimental unit is volume displaced abovethe first surface of the substrate. The method also includes samplingthe population of cells in the at least one experimental unit bycollecting the volume displaced portion of the population of cells.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface defining a first array of wells, each wellincluding an inner surface, each inner surface defining a second arrayof microwells, each microwell having an opening diameter, includessampling a population of cells in at least one microwell by applying apicking device to the first surface of the substrate. The deviceincludes at least one needle and/or nanopipette facing the firstsurface. The at least one needle and/or nanopipette has an externaldiameter less than the opening diameter of each microwell and aninternal diameter capable of accommodating a target cell diameter. Theat least one needle and/or nanopipette is inserted into at least onemicrowell holding a population of cells. The method also includeswithdrawing a sample of the population of cells in the at least onemicrowell by using pressure to pull a portion of the population of cellsfrom the at least one microwell into the device.

In an embodiment, a method for sampling a cell population in a substrateincludes a first surface defining a first array of experimental units,includes sampling a population of cells in at least one microwell byapplying a picking device to the first surface of the substrate. Thedevice includes at least one needle and/or nanopipette facing the firstsurface. The at least one needle and/or nanopipette has an externaldiameter less than the opening diameter of each microwell and aninternal diameter capable of accommodating a target cell diameter. Theat least one needle and/or nanopipette is inserted into at least oneexperimental unit holding a population of cells. The method alsoincludes withdrawing a sample of the population of cells in the at leastone experimental unit by using pressure to pull a portion of thepopulation of cells from the at least one experimental unit into thedevice.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface defining a first array of wells, each wellincluding inner surface defining a second array of microwells, includessampling a population of cells in fluid in at least one microwell byapplying focused acoustic energy to at least one microwell holding apopulation of cells in fluid. The focused acoustic energy is applied ina manner effective to eject a droplet from the at least one microwell.The droplet includes a sample of the population of cells in the at leastone microwell.

In an embodiment, a method for sampling a cell population in a substrateincluding a first surface defining a first array of experimental unitsincludes sampling a population of cells in fluid in at least oneexperimental unit by applying focused acoustic energy to at least oneexperimental unit holding a population of cells in fluid, the focusedacoustic energy being applied in a manner effective to eject a dropletfrom the at least one experimental unit. The droplet includes a sampleof the population of cells in the at least one experimental unit.

In an embodiment, a substrate includes a first surface and a secondsurface. The first surface defines a first array of wells. Each well hasan inner surface defining a second array of microwells. The substrateincludes at least a first piece including at least a portion of thefirst surface and a second piece including at least a portion of thesecond surface. The first piece and the second piece are detachablyconnected along at least a portion of a plane parallel to the firstsurface and the second surface. The plane divides the second arrays ofmicrowells. The first array and/or the second array may be substantiallyplanar. A method for sampling a cell population in at least onemicrowell includes detaching the first piece and the second piece suchthat a first portion of the population of cells in the at least onemicrowell remains attached to the first piece and a second portion ofthe population of cells in the at least one microwell remains attachedto the second piece.

In an embodiment, a substrate includes a first surface and a secondsurface. The first surface defines a first array of experimental units.The substrate includes at least a first piece including at least aportion of the first surface and a second piece including at least aportion of the second surface. The first piece and the second piece aredetachably connected along at least a portion of a plane parallel to thefirst surface and the second surface. The plane divides the first arrayof experimental units. The first array may be substantially planar. Theexperimental units may be wells. A method for sampling a cell populationin at least one experimental unit includes detaching the first piece andthe second piece such that a first portion of the population of cells inthe at least one experimental unit remains attached to the first pieceand a second portion of the population of cells in the at least oneexperimental unit remains attached to the second piece.

In an embodiment, a substrate includes a first surface defining a firstarray of wells, each well including an inner surface, each inner surfacedefining a second array of microwells. A detachable membrane is appliedto at least a portion of at least one inner surface such that a portionof the population of cells in at least one microwell attaches to thedetachable membrane. A method of sampling the population of cells in theat least one microwell includes peeling back the detachable membranesuch that the portion of the population of cells in the at least onemicrowell remains attached to the detachable membrane.

In an embodiment, a substrate includes a first surface defining a firstarray of experimental units. A detachable membrane is applied to atleast a portion of the first surface such that a portion of thepopulation of cells in at least one experimental unit attaches to thedetachable membrane. A method of sampling the population of cells in theat least one experimental unit includes peeling back the detachablemembrane such that the portion of the population of cells in the atleast one experimental unit remains attached to the detachable membrane.

In an embodiment, a substrate includes a first surface and a secondsurface. The first surface defines a first array of wells. Each well hasan inner surface, each inner surface defining a second array ofmicrochannels. Each microchannel has a first opening in the firstsurface and a second opening in the second surface. A first detachablemembrane is applied to at least a portion of at least one inner surfacesuch that at least some of the population of cells in at least onemicrochannel attach to the first detachable membrane. A seconddetachable membrane is applied to at least a portion of the secondsurface such that at least some of the population of cells in at leastone microchannel attach to the second detachable membrane. A method ofsampling the population of cells in the at least one microchannelincludes peeling back the first detachable membrane such that the atleast some of the population of cells in the at least one microchannelremain attached to the first detachable membrane and/or the seconddetachable membrane such that the at least some of the population ofcells in the at least one microchannel remain attached to the seconddetachable membrane.

In an embodiment, a substrate includes a first surface and a secondsurface. The first surface defines a first array of experimental units.Each experimental unit has a first opening in the first surface and asecond opening in the second surface. A first detachable membrane isapplied to at least a portion of the first surface such that at leastsome of the population of cells in at least one experimental unit attachto the first detachable membrane. A second detachable membrane isapplied to at least a portion of the second surface such that at leastsome of the population of cells in at least one experimental unit attachto the second detachable membrane. A method of sampling the populationof cells in the at least one experimental unit includes peeling back thefirst detachable membrane such that the at least some of the populationof cells in the at least one experimental unit remain attached to thefirst detachable membrane and/or the second detachable membrane suchthat the at least some of the population of cells in the at least oneexperimental unit remain attached to the second detachable membrane.

In an embodiment, a method for culturing cells in a sample derived froman environment includes applying the sample to the first surface of asubstrate such that at least one cell occupies at least one microwell,well, or experimental unit, applying a semi-permeable membrane to atleast a portion of the first surface such that a nutrient can diffuseinto the at least one microwell, well, or experimental unit and escapeof the at least one occupying cell from the at least one microwell,well, or experimental unit is prevented and/or mitigated, and incubatingthe at least once occupying cell in the at least one microwell, well, orexperimental unit with at least one nutrient.

In an embodiment, a method for culturing and adaptation of cells in asample derived from an environment in a substrate includes applying thesample to the first surface of the substrate such that at least one celloccupies at least one microwell, well, or experimental unit, applying asemi-permeable membrane to at least a portion of the first surface suchthat a nutrient can diffuse into the at least one microwell, well, orexperimental unit and escape of the at least one occupying cell from theat least one microwell, well, or experimental unit is prevented and/ormitigated, incubating the at least one occupying cell in the at leastone microwell, well, or experimental unit with at least one nutrient,gradually transitioning over a period of time from the at least onenutrient to at least one alternative nutrient formulation usingprogressive partial exchange, and detecting growth of the at least oneoccupying cell in the at least one microwell, well, or experimentalunit.

In some embodiments, pooled molecular assay data elements may beassigned and/or correlated back to individual locations of origin. In anembodiment, microarray includes a plurality of locations for applying asample. Each location on a microarray may be configured to receive aportion of the sample. Each location is marked with a unique tag capableof identifying from which location a portion of the sample came afterthat portion of the sample is removed from the microarray.

In an embodiment, a method of identifying from which location on amicroarray a portion of a sample comprising at least one nucleic acidmolecule came, after that portion of the sample is removed from themicroarray, includes the step of (a) applying one or more portions ofthe sample onto one or more of a plurality of locations on themicroarray. Each location is marked with a unique tag comprising anucleic acid molecule. The nucleic acid molecule includes (i) alocation-specific nucleotide sequence; and (ii) a first target-specificnucleotide sequence. The method also includes the step of (b) allowingthe nucleic acid molecule found in at least one portion of the sample toanneal to a tag marking a location. The method further includes the stepof (c) performing primer extension, reverse transcription,single-stranded ligation, or double-stranded ligation on the populationof annealed nucleic acid molecules, thereby incorporating alocation-specific nucleotide sequence into each nucleic acid moleculeproduced by primer extension, reverse transcription, single-strandedligation, or double-stranded ligation. The method further includes thesteps of (d) combining the population of nucleic acid molecules producedin step (c); (e) sequencing the population of combined nucleic acidmolecules, thereby obtaining the sequence of one or morelocation-specific nucleotide sequences; and (f) correlating the sequenceof at least one location-specific nucleotide sequence obtained from thepopulation of combined nucleic acid molecules to the location on themicroarray marked with a tag including the location-specific nucleotidesequence, thereby identifying from which location on a microarray aportion of a sample comprising at least one nucleic acid molecule came.The sample may include at least one cell, and the cell may replicateafter step (a) and before step (b). A portion of the portion of thesample may be removed from at least one location before step (b), andsaid portion of the portion of the sample may be stored in a separatereceptacle correlated to the original location of the portion of thesample on the microarray.

In an embodiment, a method of manufacturing a microarray with aplurality of locations for applying a sample, wherein at least onelocation is marked with a unique tag, includes the step of (a)synthesizing a plurality of tags. Each tag includes a nucleic acidmolecule including: (i) a location-specific nucleotide sequence; and(ii) a first target-specific nucleotide sequence. The method furthercomprises the step of (b) placing a tag on at least one location of theplurality of locations on the microarray. A unique tag may include anucleic acid molecule including (i) a location-specific nucleotidesequence; and (ii) a first target-specific nucleotide sequence. The tagfurther may include an amplification primer binding site and/or anadapter nucleotide sequence.

A microfabricated device may be manufactured, via injection molding,using cyclic olefin polymer. The substrate or chip may have asubstantially planar surface with dimensions of about 1 inch by about 3inches. The surface further may define about 200,000 wells, each wellhaving a diameter of about 50 μm. Alternatively, the surface may defineabout 800,000 microwells, each microwell having a diameter of about 25μm. The surface may be treated with a corona plasma treatment to make itmore hydrophilic.

Microorganisms (e.g., bacteria) isolated from soil may be diluted intosoil extract nutrient and applied to the surface of the chip. Next, amembrane may be applied to the surface of the chip. The membrane may be,for example, a hydrogel layer. The membrane may be reversibly orirreversibly attached or affixed to the chip using, for example,lamination. Then, a second device may be placed on first of the membraneand surface of the chip to divide the surface of the chip intopartitions. Each partition of the surface has some subset of the wellsor microwells therein.

In an on-chip adaptation, each partition may be loaded with anenvironmentally-derived nutrient media. During an incubation period, thechip may be monitored to observe bacterial growth using, for example, anoptical system to detect differences as bacteria grow in microwells.Over time, the nutrient media may be slowly adjusted until it is a fullyformulated media that can be used to grow the bacteria in a laboratoryenvironment.

In an off-chip adaptation, any wells or microwells where bacterialgrowth is observed may be sampled. The bacteria samples may betransferred to, for example, a plate, second chip, or a traditional toollike a petri dish for adaptation to formulated media.

Some embodiments may be used for optimizing media. For example, a singlespecies of bacteria may be applied to a surface of the chip. Next, amembrane may be applied to the surface of the chip. The membrane may be,for example, a hydrogel layer. The membrane may be reversibly orirreversibly connected to or affixed to the chip using, for example,lamination. Then, a second device may be placed on top of the membraneand/or surface of the chip to divide the membrane and/or surface of thechip into partitions. Each partition of the membrane and/or surface hassome subset of the wells or microwells underlying the membrane and/orthereon the surface. Each partition may be loaded with a differentmedium (e.g., derived from a different environment). During anincubation period, the chip may be monitored to observe growth rates inthe wells or microwells with different nutrient formulations.

Some embodiments may be used for screening for microbes that solubilizephosphate, particularly for agricultural applications. Phosphorus is amajor essential macronutrient for plants. Soil phosphorus may be managedto optimize crop production and is applied to soil in the form ofphosphate fertilizers. However, a large portion of soluble inorganicphosphate, which is applied to the soil as chemical fertilizer, isimmobilized rapidly and becomes unavailable to plants. Phosphatesolubilizing bacteria strains hydrolyze organic and inorganic phosphorusfrom insoluble compounds, and may be used to improve plant growth andyield. A sample that includes microbes may be derived from anenvironment, such as soil (particularly if organic and inorganicphosphorus are being effectively hydrolyzed from insoluble compounds inthe environment). According to an embodiment, the sample may be loadedonto a surface of a substrate with at least one array of experimentalunits such that at least some of the microbes occupy a plurality of theexperimental units. The occupying microbes may be incubated in theplurality of experimental units with different forms of phosphate andcompared/screened for their ability to hydrolyze the different forms ofphosphate.

Some embodiments may be used for screening for mutants that have highergrowth rates. A sample of microorganisms (e.g., bacteria) may be derivedfrom a single lab culture that has been treated with a mutagen.According to an embodiment, the sample may be loaded onto a surface of asubstrate with at least one array of experimental units such that atleast some of the bacteria occupy a plurality of the experimental units.The occupying bacteria may be incubated in the plurality of experimentalunits with at least one nutrient and observed/compared to identify theexperimental unit(s) with higher growth rates.

Some embodiments may be used for identifying, for example, novelantibiotics, antifungals, or anti-viral compounds. A sample thatincludes microorganisms (e.g., bacteria) may be derived from anenvironment. According to an embodiment, the sample may be loaded onto asurface of a substrate with at least one array of experimental unitssuch that at least some of the bacteria occupy a plurality of theexperimental units. The occupying bacteria are grown in the plurality ofexperimental units for a period of time, then a layer of indicatorbacteria is grown on first of the occupying bacteria (this may require athin layer of agar). Experimental units that produce clear plaques inthe layer of indicator bacteria indicate antibiotic production.

Some embodiments may be used for screening for a particular enzymeactivity. According to an embodiment, a sample including cells may beloaded onto a surface of a substrate with at least one array ofexperimental units such that at least one of the cells occupies aplurality of the experimental units. The occupying cells are grown inthe plurality of experimental units for a period of time, then asubstrate that produces a color when cleaved by the enzyme is added.Experimental units that produce this color reaction indicate the desiredenzyme activity.

Some embodiments may be used for screening for microorganisms thatperform bioremediation, particularly for environmental clean-upapplications. Pollution and environmental contamination is complex. Forexample, the effects of an oil spill or other release of liquidpetroleum hydrocarbon into an environment depends upon many factors. Ina marine environment, the type(s) of oil, the temperature(s) of thewater, and the type(s) of shoreline all affect cleanup. A sample thatincludes microorganisms may be derived from an environment, such asoil-bearing rock formations. According to an embodiment, the sample maybe loaded onto a surface of a substrate with at least one array ofexperimental units such that at least some of the microorganisms occupya plurality of the experimental units. The occupying microorganisms maybe incubated in the plurality of experimental units with different typesof oil and compared/screened for their ability to remove or neutralizethe oil.

CONCLUSION

While various inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

Also, various inventive concepts may be embodied as one or more methods,of which an example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” “defining” and thelike are to be understood to be open-ended, i.e., to mean including butnot limited to. Only the transitional phrases “consisting of” and“consisting essentially of” shall be closed or semi-closed transitionalphrases, respectively, as set forth in the United States Patent OfficeManual of Patent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of screening for at least one biologicalentity of interest in a sample using a microfabricated device having atop surface defining an array of microwells, the method comprising:loading into at least one microwell of the array of microwells: (a) atleast one cell from the sample; (b) an indicator substance; and (c) anamount of a nutrient; applying a membrane to the microfabricated deviceto retain the at least one cell in the at least one microwell;incubating the microfabricated device at predetermined conditions for aduration of time to grow a plurality of cells from the at least one cellin the at least one microwell; evaluating an optical property of the atleast one microwell; and determining a presence or absence of at leastone biological entity of interest in the at least one microwell based onthe optical property.
 2. The method of claim 1, wherein the opticalproperty of the at least one microwell correlates to the amount or formof the indicator substance.
 3. The method of claim 1, wherein theindicator substance is an inorganic phosphate salt that is insoluble inwater.
 4. The method of claim 3, wherein at least one biological entityof interest comprises a microorganism that solubilizes the inorganicphosphate salt.
 5. The method of claim 1, wherein the indicatorcomprises a pH sensitive dye.
 6. The method of claim 5, wherein the pHsensitive dye can change color in a visible light range within aselected range of pH.
 7. The method of claim 5, wherein the pH sensitivedye is fluorescent in a first range of pH and not fluorescent in asecond, different range of pH.
 8. The method of claim 5, wherein the pHsensitive dye is a fluorescent dye whose fluorescence changes atdifferent pH.
 9. The method of claim 1, wherein the at least onebiological entity of interest comprises a microorganism that produces anacid.
 10. The method of claim 1, wherein the at least one biologicalentity of interest comprises a eukaryotic cell.
 11. The method of claim1, wherein the at least one biological entity of interest comprisesbacteria.
 12. The method of claim 1, wherein measuring the opticalproperty comprises taking a plurality of measurements of the opticalproperty at different times during the course of the incubation.
 13. Themethod of claim 1, further comprising: if a biological entity ofinterest is determined to be present in the at least one microwell,transferring at least some of the plurality of cells after incubation toa target location.
 14. The method of claim 1, wherein the at least onemicrowell includes a plurality of microwells, and wherein loading the atleast one cell comprises loading into each of the plurality ofmicrowells, on average, one cell.
 15. The method of claim 1, whereineach microwell of the array of microwells has a diameter of about 25 μmto about 500 μm.
 16. The method of claim 1, wherein the surface densityof the array of microwells is at least 750 microwells per cm².
 17. Themethod of claim 1, wherein evaluating the optical property comprises:(a) measuring the optical property of the at least one microwell afterthe at least one cell, the indicator substance, and the nutrient havebeen loaded and before incubation; (b) measuring the optical property ofthe at least one microwell after incubation; and (c) comparing themeasured optical property before incubation and after incubation.
 18. Amethod of screening for at least one biological entity of interest in asample using a microfabricated device having a top surface defining anarray of microwells, the method comprising: loading into at least onemicrowell of the array of microwells: (a) at least one cell from thesample; (b) an indicator substance; and (c) an amount of a nutrient;applying a membrane to the microfabricated device to retain the at leastone cell in the at least one microwell; incubating the microfabricateddevice at predetermined conditions for a duration of time to grow aplurality of cells from the at least one cell in the at least onemicrowell; and determining a presence or absence of at least onebiological entity of interest in the at least one microwell based on achange of the indicator substance after incubation.
 19. A method ofscreening for at least one biological entity of interest in a sampleusing a microfabricated device having a top surface defining an array ofmicrowells, wherein each of the microwells has a bottom, the methodcomprising: depositing an indicator substance into each of a pluralityof microwells of the array of microwells; loading the sample onto themicrofabricated device such that at least one microwell of the pluralityof microwells receives at least one cell from the sample and an amountof a nutrient; applying a membrane to the microfabricated device toretain the received at least one cell and the nutrient in each of theplurality of microwells; incubating the microfabricated device atpredetermined conditions for a duration of time; and determining apresence or absence of at least one biological entity of interest in theat least one microwell of the plurality of microwells based on ameasurement of an optical property of the at least one microwell.